Microfluidic devices for optically-driven convection and displacement, kits and methods thereof

ABSTRACT

Apparatuses and methods are described for the use of optically driven bubble, convective and displacing fluidic flow to provide motive force in microfluidic devices. Alternative motive modalities are useful to selectively dislodge and displace micro-objects, including biological cells, from a variety of locations within the enclosure of a microfluidic device.

This application is a non-provisional application claiming the benefitunder 35 U.S.C. 119(e) of U.S. Provisional Application No. 62/273,104,filed on Dec. 30, 2015; U.S. Provisional Application No. 62/314,889,filed on Mar. 29, 2016; and of U.S. Provisional Application No.62/428,539, filed on Dec. 1, 2016, each of which disclosures is hereinincorporated by reference in its entirety.

BACKGROUND

As the field of microfluidics develops, microfluidic devices have becomeconvenient platforms for processing and manipulating micro-objects suchas biological cells. Some embodiments of the present invention aredirected to methods and devices for the use of optically driven bubble,convective and displacing fluidic flow to provide motive force inmicrofluidic devices.

SUMMARY

In one aspect, a microfluidic device is provided, where the microfluidicdevice includes an enclosure having a flow region and a sequestrationpen, where the sequestration pen includes: a connection region, anisolation region and a displacement force generation region, where: theconnection region includes a proximal opening to the flow region and adistal opening to the isolation region; and the isolation regionincludes at least one fluidic connection to the displacement forcegeneration region; and the displacement force generation region furtherincludes a thermal target.

In another aspect, a microfluidic device is provided, where themicrofluidic device includes an enclosure having a microfluidic circuitconfigured to contain a fluidic medium, where the microfluidic circuitis configured to accommodate at least one cyclic flow of the fluidicmedium; and a first thermal target disposed on a surface of theenclosure within the microfluidic circuit, where the first thermaltarget is configured to produce a first cyclic flow of the fluidicmedium upon optical illumination.

In yet another aspect, a microfluidic device is provided, where themicrofluidic device includes an enclosure having a microfluidic channeland a sequestration pen, and further where the sequestration pen isadjacent to and opens off of the microfluidic channel and a thermaltarget is disposed in the channel adjacent to an opening to asequestration pen, and wherein the thermal target is further configuredto direct a flow of the fluidic medium into the sequestration pen uponoptical illumination.

In another aspect, a kit for culturing micro-objects is provided, wherethe kit includes: a microfluidic device as described herein; and, one ormore reagents configured to provide at least one coated surface withinan enclosure of the microfluidic device.

In another aspect, a method is provided for dislodging one or moremicro-objects within a microfluidic device, the method including thesteps of: illuminating a selected discrete region containing or adjacentto one or more micro-objects disposed within a fluidic medium in anenclosure of the microfluidic device, wherein the enclosure includes amicrofluidic circuit including a flow region and a substrate; andmaintaining the illumination of the selected discrete region of a firstperiod of time sufficient to generate a dislodging force, dislodging theone or more micro-objects from the surface.

In yet another aspect, a method is provided for mixing fluidic media,and/or micro-objects contained therein, within an enclosure of amicrofluidic device, the method including: focusing a light source on athermal target disposed on a surface of the enclosure within amicrofluidic circuit including at least one fluidic medium and/ormicro-objects, thereby heating a first portion of the at least onefluidic medium; and inducing a cyclic flow of the at least one fluidicmedium within the microfluidic circuit thereby mixing the fluidic mediaand/or micro-objects disposed therein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graphical representation of a system for use with amicrofluidic device and associated control equipment according to someembodiments of the disclosure.

FIGS. 1B and 1C are graphical representations of a microfluidic deviceaccording to some embodiments of the disclosure.

FIGS. 2A and 2B are graphical representations of isolation pensaccording to some embodiments of the disclosure.

FIG. 2C is a graphical representation of a detailed sequestration penaccording to some embodiments of the disclosure.

FIGS. 2D-F are graphical representations of sequestration pens accordingto some other embodiments of the disclosure.

FIG. 2G is a graphical representation of a microfluidic device accordingto an embodiment of the disclosure.

FIG. 2H is a graphical representation of a coated surface of themicrofluidic device according to an embodiment of the disclosure.

FIG. 3A is a graphical representation of a specific example of a systemfor use with a microfluidic device and associated control equipmentaccording to some embodiments of the disclosure.

FIG. 3B is a schematic representation an imaging device according tosome embodiments of the disclosure.

FIGS. 4A-4I are graphical representations of various thermal targetsaccording to embodiments of the disclosure.

FIGS. 5A-5E are graphical representations of sequestration pensaccording to some embodiments of the disclosure.

FIGS. 6A-6D are graphical representations of sequestration pensaccording to some embodiments of the disclosure.

FIGS. 7A-7F are graphical representations of further embodiments ofsequestration pens according to the disclosure.

FIGS. 8A-8D illustrate microfluidic devices according to someembodiments of the disclosure.

FIGS. 9A-9D are photographic representations of the use ofoptically-driven forces used to export cells from a sequestration penaccording to some embodiments of the disclosure, and viabilitythereafter.

FIGS. 10A-10C depict the use of optically driven displacement to exportcells from a sequestration pen and viability thereafter.

FIGS. 11A-11C are photographic representations of cells maintained in amicrofluidic device before and after optically driven displacementaccording to the disclosure.

FIG. 12A-12C are photographic representations of cells maintained in amicrofluidic device before and after optically driven displacementaccording to the disclosure.

FIGS. 13A-13C are photographic representations of a method usingillumination to create a cyclized flow capable of moving micro-objects.

FIGS. 14A-14C are photographic representations of one embodiment of themethod of laser illumination for dislodging micro-objects.

FIGS. 15A-15E are photographic representations of another embodiment ofthe method of laser illumination for dislodging micro-objects.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

This specification describes exemplary embodiments and applications ofthe disclosure. The disclosure, however, is not limited to theseexemplary embodiments and applications or to the manner in which theexemplary embodiments and applications operate or are described herein.Moreover, the figures may show simplified or partial views, and thedimensions of elements in the figures may be exaggerated or otherwisenot in proportion. In addition, as the terms “on,” “attached to,”“connected to,” “coupled to,” or similar words are used herein, oneelement (e.g., a material, a layer, a substrate, etc.) can be “on,”“attached to,” “connected to,” or “coupled to” another elementregardless of whether the one element is directly on, attached to,connected to, or coupled to the other element or there are one or moreintervening elements between the one element and the other element.Also, unless the context dictates otherwise, directions (e.g., above,below, top, bottom, side, up, down, under, over, upper, lower,horizontal, vertical, “x,” “y,” “z,” etc.), if provided, are relativeand provided solely by way of example and for ease of illustration anddiscussion and not by way of limitation. In addition, where reference ismade to a list of elements (e.g., elements a, b, c), such reference isintended to include any one of the listed elements by itself, anycombination of less than all of the listed elements, and/or acombination of all of the listed elements. Section divisions in thespecification are for ease of review only and do not limit anycombination of elements discussed.

Where dimensions of microfluidic features are described as having awidth or an area, the dimension typically is described relative to anx-axial and/or y-axial dimension, both of which lie within a plane thatis parallel to the substrate and/or cover of the microfluidic device.The height of a microfluidic feature may be described relative to az-axial direction, which is perpendicular to a plane that is parallel tothe substrate and/or cover of the microfluidic device. In someinstances, a cross sectional area of a microfluidic feature, such as achannel or a passageway, may be in reference to a x-axial/z-axial, ay-axial/z-axial, or an x-axial/y-axial area.

As used herein, “substantially” means sufficient to work for theintended purpose. The term “substantially” thus allows for minor,insignificant variations from an absolute or perfect state, dimension,measurement, result, or the like such as would be expected by a personof ordinary skill in the field but that do not appreciably affectoverall performance. When used with respect to numerical values orparameters or characteristics that can be expressed as numerical values,“substantially” means within ten percent.

The term “ones” means more than one.

As used herein, the term “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10,or more.

As used herein, the term “disposed” encompasses within its meaning“located.”

As used herein, a “microfluidic device” or “microfluidic apparatus” is adevice that includes one or more discrete microfluidic circuitsconfigured to hold a fluid, each microfluidic circuit comprised offluidically interconnected circuit elements, including but not limitedto region(s), flow path(s), channel(s), chamber(s), and/or pen(s), andat least one port configured to allow the fluid (and, optionally,micro-objects suspended in the fluid) to flow into and/or out of themicrofluidic device. Typically, a microfluidic circuit of a microfluidicdevice will include a flow region, which may include a microfluidicchannel, and at least one chamber, and will hold a volume of fluid ofless than about 1 mL, e.g., less than about 750, 500, 250, 200, 150,100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 microliters. Incertain embodiments, the microfluidic circuit holds about 1-2, 1-3, 1-4,1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50,10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300microliters. The microfluidic circuit may be configured to have a firstend fluidically connected with a first port (e.g., an inlet) in themicrofluidic device and a second end fluidically connected with a secondport (e.g., an outlet) in the microfluidic device.

As used herein, a “nanofluidic device” or “nanofluidic apparatus” is atype of microfluidic device having a microfluidic circuit that containsat least one circuit element configured to hold a volume of fluid ofless than about 1 microliter, e.g., less than about 750, 500, 250, 200,150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less.A nanofluidic device may comprise a plurality of circuit elements (e.g.,at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200,250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). Incertain embodiments, one or more (e.g., all) of the at least one circuitelements is configured to hold a volume of fluid of about 100 pL to 1nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, one or more (e.g.,all) of the at least one circuit elements are configured to hold avolume of fluid of about 20 nL to 200 nL, 100 to 200 nL, 100 to 300 nL,100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to600 nL, or 250 to 750 nL.

A “microfluidic channel” or “flow channel” as used herein refers to flowregion of a microfluidic device having a length that is significantlylonger than both the horizontal and vertical dimensions. For example,the flow channel can be at least 5 times the length of either thehorizontal or vertical dimension, e.g., at least 10 times the length, atleast 25 times the length, at least 100 times the length, at least 200times the length, at least 500 times the length, at least 1,000 timesthe length, at least 5,000 times the length, or longer. In someembodiments, the length of a flow channel is in the range of from about100,000 microns to about 500,000 microns, including any rangetherebetween. In some embodiments, the horizontal dimension is in therange of from about 100 microns to about 1000 microns (e.g., about 150to about 500 microns) and the vertical dimension is in the range of fromabout 25 microns to about 200 microns, e.g., from about 40 to about 150microns. It is noted that a flow channel may have a variety of differentspatial configurations in a microfluidic device, and thus is notrestricted to a perfectly linear element. For example, a flow channelmay be, or include one or more sections having, the followingconfigurations: curve, bend, spiral, incline, decline, fork (e.g.,multiple different flow paths), and any combination thereof. Inaddition, a flow channel may have different cross-sectional areas alongits path, widening and constricting to provide a desired fluid flowtherein. The flow channel may include valves, and the valves may be ofany type known in the art of microfluidics. Examples of microfluidicchannels that include valves are disclosed in U.S. Pat. Nos. 6,408,878and 9,227,200, each of which is herein incorporated by reference in itsentirety.

As used herein, the term “obstruction” refers generally to a bump orsimilar type of structure that is sufficiently large so as to partially(but not completely) impede movement of target micro-objects between twodifferent regions or circuit elements in a microfluidic device. The twodifferent regions/circuit elements can be, for example, the connectionregion and the isolation region of a microfluidic sequestration pen.

As used herein, the term “constriction” refers generally to a narrowingof a width of a circuit element (or an interface between two circuitelements) in a microfluidic device. The constriction can be located, forexample, at the interface between the isolation region and theconnection region of a microfluidic sequestration pen of the instantdisclosure.

As used herein, the term “transparent” refers to a material which allowsvisible light to pass through without substantially altering the lightas is passes through.

As used herein, the term “micro-object” refers generally to anymicroscopic object that may be isolated and/or manipulated in accordancewith the present disclosure. Non-limiting examples of micro-objectsinclude: inanimate micro-objects such as microparticles; microbeads(e.g., polystyrene beads, Luminex™ beads, or the like); magnetic beads;microrods; microwires; quantum dots, and the like; biologicalmicro-objects such as cells; biological organelles; vesicles, orcomplexes; synthetic vesicles; liposomes (e.g., synthetic or derivedfrom membrane preparations); lipid nanorafts, and the like; or acombination of inanimate micro-objects and biological micro-objects(e.g., microbeads attached to cells, liposome-coated micro-beads,liposome-coated magnetic beads, or the like). Beads may includemoieties/molecules covalently or non-covalently attached, such asfluorescent labels, proteins, carbohydrates, antigens, small moleculesignaling moieties, or other chemical/biological species capable of usein an assay. Lipid nanorafts have been described, for example, inRitchie et al. (2009) “Reconstitution of Membrane Proteins inPhospholipid Bilayer Nanodiscs,” Methods Enzymol., 464:211-231.

As used herein, the term “cell” is used interchangeably with the term“biological cell.” Non-limiting examples of biological cells includeeukaryotic cells, plant cells, animal cells, such as mammalian cells,reptilian cells, avian cells, fish cells, or the like, prokaryoticcells, bacterial cells, fungal cells, protozoan cells, or the like,cells dissociated from a tissue, such as muscle, cartilage, fat, skin,liver, lung, neural tissue, and the like, immunological cells, such as Tcells, B cells, natural killer cells, macrophages, and the like, embryos(e.g., zygotes), oocytes, ova, sperm cells, hybridomas, cultured cells,cells from a cell line, cancer cells, infected cells, transfected and/ortransformed cells, reporter cells, and the like. A mammalian cell canbe, for example, from a human, a mouse, a rat, a horse, a goat, a sheep,a cow, a primate, or the like.

A colony of biological cells is “clonal” if all of the living cells inthe colony that are capable of reproducing are daughter cells derivedfrom a single parent cell. In certain embodiments, all the daughtercells in a clonal colony are derived from the single parent cell by nomore than 10 divisions. In other embodiments, all the daughter cells ina clonal colony are derived from the single parent cell by no more than14 divisions. In other embodiments, all the daughter cells in a clonalcolony are derived from the single parent cell by no more than 17divisions. In other embodiments, all the daughter cells in a clonalcolony are derived from the single parent cell by no more than 20divisions. The term “clonal cells” refers to cells of the same clonalcolony.

As used herein, a “colony” of biological cells refers to 2 or more cells(e.g. about 2 to about 20, about 4 to about 40, about 6 to about 60,about 8 to about 80, about 10 to about 100, about 20 to about 200, about40 to about 400, about 60 to about 600, about 80 to about 800, about 100to about 1000, or greater than 1000 cells).

As used herein, the term “maintaining (a) cell(s)” refers to providingan environment comprising both fluidic and gaseous components and,optionally a surface, that provides the conditions necessary to keep thecells viable and/or expanding.

As used herein, the term “expanding” when referring to cells, refers toincreasing in cell number.

A “component” of a fluidic medium is any chemical or biochemicalmolecule present in the medium, including solvent molecules, ions, smallmolecules, antibiotics, nucleotides and nucleosides, nucleic acids,amino acids, peptides, proteins, sugars, carbohydrates, lipids, fattyacids, cholesterol, metabolites, or the like.

As used herein in reference to a fluidic medium, “diffuse” and“diffusion” refer to thermodynamic movement of a component of thefluidic medium down a concentration gradient.

The phrase “flow of a medium” means bulk movement of a fluidic mediumprimarily due to any mechanism other than diffusion. For example, flowof a medium can involve movement of the fluidic medium from one point toanother point due to a pressure differential between the points. Suchflow can include a continuous, pulsed, periodic, random, intermittent,or reciprocating flow of the liquid, or any combination thereof. Whenone fluidic medium flows into another fluidic medium, turbulence andmixing of the media can result.

The phrase “substantially no flow” refers to a rate of flow of a fluidicmedium that, averaged over time, is less than the rate of diffusion ofcomponents of a material (e.g., an analyte of interest) into or withinthe fluidic medium. The rate of diffusion of components of such amaterial can depend on, for example, temperature, the size of thecomponents, and the strength of interactions between the components andthe fluidic medium.

As used herein in reference to different regions within a microfluidicdevice, the phrase “fluidically connected” means that, when thedifferent regions are substantially filled with fluid, such as fluidicmedia, the fluid in each of the regions is connected so as to form asingle body of fluid. This does not mean that the fluids (or fluidicmedia) in the different regions are necessarily identical incomposition. Rather, the fluids in different fluidically connectedregions of a microfluidic device can have different compositions (e.g.,different concentrations of solutes, such as proteins, carbohydrates,ions, or other molecules) which are in flux as solutes move down theirrespective concentration gradients and/or fluids flow through themicrofluidic device.

As used herein, a “flow path” refers to one or more fluidicallyconnected circuit elements (e.g. channel(s), region(s), chamber(s) andthe like) that define, and are subject to, the trajectory of a flow ofmedium. A flow path is thus an example of a swept region of amicrofluidic device. Other circuit elements (e.g., unswept regions) maybe fluidically connected with the circuit elements that comprise theflow path without being subject to the flow of medium in the flow path.

As used herein, “isolating a micro-object” confines a micro-object to adefined area within the microfluidic device. The micro-object may stillbe capable of motion within an in situ-generated capture structure.

A microfluidic (or nanofluidic) device can comprise “swept” regions and“unswept” regions. As used herein, a “swept” region is comprised of oneor more fluidically interconnected circuit elements of a microfluidiccircuit, each of which experiences a flow of medium when fluid isflowing through the microfluidic circuit. The circuit elements of aswept region can include, for example, regions, channels, and all orparts of chambers. As used herein, an “unswept” region is comprised ofone or more fluidically interconnected circuit element of a microfluidiccircuit, each of which experiences substantially no flux of fluid whenfluid is flowing through the microfluidic circuit. An unswept region canbe fluidically connected to a swept region, provided the fluidicconnections are structured to enable diffusion but substantially no flowof media between the swept region and the unswept region. Themicrofluidic device can thus be structured to substantially isolate anunswept region from a flow of medium in a swept region, while enablingsubstantially only diffusive fluidic communication between the sweptregion and the unswept region. For example, a flow channel of amicro-fluidic device is an example of a swept region while an isolationregion (described in further detail below) of a microfluidic device isan example of an unswept region.

As used herein, a “sacrificial feature” refers to a microfluidic circuitelement which may be used as a thermal target in the microfluidicdevices and methods of the disclosure, and which is at least partiallydestroyed upon being sufficiently illuminated so as to generate abubble, a cavitating force, or a shear flow as described herein.

The capability of biological micro-objects (e.g., biological cells) toproduce specific biological materials (e.g., proteins, such asantibodies) can be assayed in such a microfluidic device. In a specificembodiment of an assay, sample material comprising biologicalmicro-objects (e.g., cells) to be assayed for production of an analyteof interest can be loaded into a swept region of the microfluidicdevice. Ones of the biological micro-objects (e.g., mammalian cells,such as human cells) can be selected for particular characteristics anddisposed in unswept regions. The remaining sample material can then beflowed out of the swept region and an assay material flowed into theswept region. Because the selected biological micro-objects are inunswept regions, the selected biological micro-objects are notsubstantially affected by the flowing out of the remaining samplematerial or the flowing in of the assay material. The selectedbiological micro-objects can be allowed to produce the analyte ofinterest, which can diffuse from the unswept regions into the sweptregion, where the analyte of interest can react with the assay materialto produce localized detectable reactions, each of which can becorrelated to a particular unswept region. Any unswept region associatedwith a detected reaction can be analyzed to determine which, if any, ofthe biological micro-objects in the unswept region are sufficientproducers of the analyte of interest.

Microfluidic devices and systems for operating and observing suchdevices. FIG. 1A illustrates an example of a microfluidic device 100 anda system 150 which can be used for generation of embryos in vitro,including selecting and evaluating ova and/or oocytes and/or sperm. Aperspective view of the microfluidic device 100 is shown having apartial cut-away of its cover 110 to provide a partial view into themicrofluidic device 100. The microfluidic device 100 generally includesa microfluidic circuit 120 comprising a flow path 106 through which afluidic medium 180 can flow, optionally carrying one or moremicro-objects (not shown) into and/or through the microfluidic circuit120. Although a single microfluidic circuit 120 is illustrated in FIG.1A, suitable microfluidic devices can include a plurality (e.g., 2 or 3)of such microfluidic circuits. Regardless, the microfluidic device 100can be configured to be a nanofluidic device. As illustrated in FIG. 1A,the microfluidic circuit 120 may include a plurality of microfluidicsequestration pens 124, 126, 128, and 130, where each sequestration pensmay have one or more openings in fluidic communication with flow path106. In some embodiments of the device of FIG. 1A, the sequestrationpens may have only a single opening in fluidic communication with theflow path 106. As discussed further below, the microfluidicsequestration pens include various features and structures that havebeen optimized for retaining micro-objects in the microfluidic device,such as microfluidic device 100, even when a medium 180 is flowingthrough the flow path 106. Before turning to the foregoing, however, abrief description of microfluidic device 100 and system 150 is provided.

As generally illustrated in FIG. 1A, the microfluidic circuit 120 isdefined by an enclosure 102. Although the enclosure 102 can bephysically structured in different configurations, in the example shownin FIG. 1A the enclosure 102 is depicted as including a supportstructure 104 (e.g., a base), a microfluidic circuit structure 108, anda cover 110. The support structure 104, microfluidic circuit structure108, and cover 110 can be attached to each other. For example, themicrofluidic circuit structure 108 can be disposed on an inner surface109 of the support structure 104, and the cover 110 can be disposed overthe microfluidic circuit structure 108. Together with the supportstructure 104 and cover 110, the microfluidic circuit structure 108 candefine the elements of the microfluidic circuit 120.

The support structure 104 can be at the bottom and the cover 110 at thetop of the microfluidic circuit 120 as illustrated in FIG. 1A.Alternatively, the support structure 104 and the cover 110 can beconfigured in other orientations. For example, the support structure 104can be at the top and the cover 110 at the bottom of the microfluidiccircuit 120. Regardless, there can be one or more ports 107 eachincluding a passage into or out of the enclosure 102. Examples of apassage include a valve, a gate, a pass-through hole, or the like. Asillustrated, port 107 is a pass-through hole created by a gap in themicrofluidic circuit structure 108. However, the port 107 can besituated in other components of the enclosure 102, such as the cover110. Only one port 107 is illustrated in FIG. 1A but the microfluidiccircuit 120 can have two or more ports 107. For example, there can be afirst port 107 that functions as an inlet for fluid entering themicrofluidic circuit 120, and there can be a second port 107 thatfunctions as an outlet for fluid exiting the microfluidic circuit 120.Whether a port 107 function as an inlet or an outlet can depend upon thedirection that fluid flows through flow path 106.

The support structure 104 can include one or more electrodes (not shown)and a substrate or a plurality of interconnected substrates. Forexample, the support structure 104 can include one or more semiconductorsubstrates, each of which is electrically connected to an electrode(e.g., all or a subset of the semiconductor substrates can beelectrically connected to a single electrode). The support structure 104can further include a printed circuit board assembly (“PCBA”). Forexample, the semiconductor substrate(s) can be mounted on a PCBA.

The microfluidic circuit structure 108 can define circuit elements ofthe microfluidic circuit 120. Such circuit elements can include spacesor regions that can be fluidly interconnected when microfluidic circuit120 is filled with fluid, such as flow regions (which may include or beone or more flow channels), chambers, pens, traps, and the like. In themicrofluidic circuit 120 illustrated in FIG. 1A, the microfluidiccircuit structure 108 includes a frame 114 and a microfluidic circuitmaterial 116. The frame 114 can partially or completely enclose themicrofluidic circuit material 116. The frame 114 can be, for example, arelatively rigid structure substantially surrounding the microfluidiccircuit material 116. For example, the frame 114 can comprise a metalmaterial.

The microfluidic circuit material 116 can be patterned with cavities orthe like to define circuit elements and interconnections of themicrofluidic circuit 120. The microfluidic circuit material 116 cancomprise a flexible material, such as a flexible polymer (e.g. rubber,plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or thelike), which can be gas permeable. Other examples of materials that cancompose microfluidic circuit material 116 include molded glass, anetchable material such as silicone (e.g. photo-patternable silicone or“PPS”), photo-resist (e.g., SU8), or the like. In some embodiments, suchmaterials—and thus the microfluidic circuit material 116—can be rigidand/or substantially impermeable to gas. Regardless, microfluidiccircuit material 116 can be disposed on the support structure 104 andinside the frame 114.

The cover 110 can be an integral part of the frame 114 and/or themicrofluidic circuit material 116. Alternatively, the cover 110 can be astructurally distinct element, as illustrated in FIG. 1A. The cover 110can comprise the same or different materials than the frame 114 and/orthe microfluidic circuit material 116. Similarly, the support structure104 can be a separate structure from the frame 114 or microfluidiccircuit material 116 as illustrated, or an integral part of the frame114 or microfluidic circuit material 116. Likewise, the frame 114 andmicrofluidic circuit material 116 can be separate structures as shown inFIG. 1A or integral portions of the same structure.

In some embodiments, the cover 110 can comprise a rigid material. Therigid material may be glass or a material with similar properties. Insome embodiments, the cover 110 can comprise a deformable material. Thedeformable material can be a polymer, such as PDMS. In some embodiments,the cover 110 can comprise both rigid and deformable materials. Forexample, one or more portions of cover 110 (e.g., one or more portionspositioned over sequestration pens 124, 126, 128, 130) can comprise adeformable material that interfaces with rigid materials of the cover110. In some embodiments, the cover 110 can further include one or moreelectrodes. The one or more electrodes can comprise a conductive oxide,such as indium-tin-oxide (ITO), which may be coated on glass or asimilarly insulating material. Alternatively, the one or more electrodescan be flexible electrodes, such as single-walled nanotubes,multi-walled nanotubes, nanowires, clusters of electrically conductivenanoparticles, or combinations thereof, embedded in a deformablematerial, such as a polymer (e.g., PDMS). Flexible electrodes that canbe used in microfluidic devices have been described, for example, inU.S. 2012/0325665 (Chiou et al.), the contents of which are incorporatedherein by reference. In some embodiments, the cover 110 can be modified(e.g., by conditioning all or part of a surface that faces inward towardthe microfluidic circuit 120) to support cell adhesion, viability and/orgrowth. The modification may include a coating of a synthetic or naturalpolymer. In some embodiments, the cover 110 and/or the support structure104 can be transparent to light. The cover 110 may also include at leastone material that is gas permeable (e.g., PDMS or PPS).

FIG. 1A also shows a system 150 for operating and controllingmicrofluidic devices, such as microfluidic device 100. System 150includes an electrical power source 192, an imaging device 194(incorporated within imaging module 164, where device 194 is notillustrated in FIG. 1A, per se), and a tilting device 190 (part oftilting module 166, where device 190 is not illustrated in FIG. 1A).

The electrical power source 192 can provide electric power to themicrofluidic device 100 and/or tilting device 190, providing biasingvoltages or currents as needed. The electrical power source 192 can, forexample, include one or more alternating current (AC) and/or directcurrent (DC) voltage or current sources. The imaging device 194 (part ofimaging module 164, discussed below) can comprise a device, such as adigital camera, for capturing images inside microfluidic circuit 120. Insome instances, the imaging device 194 further comprises a detectorhaving a fast frame rate and/or high sensitivity (e.g. for low lightapplications). The imaging device 194 can also include a mechanism fordirecting stimulating radiation and/or light beams into the microfluidiccircuit 120 and collecting radiation and/or light beams reflected oremitted from the microfluidic circuit 120 (or micro-objects containedtherein). The emitted light beams may be in the visible spectrum andmay, e.g., include fluorescent emissions. The reflected light beams mayinclude reflected emissions originating from an LED or a wide spectrumlamp, such as a mercury lamp (e.g. a high pressure mercury lamp) or aXenon arc lamp. As discussed with respect to FIG. 3B, the imaging device194 may further include a microscope (or an optical train), which may ormay not include an eyepiece.

System 150 further comprises a tilting device 190 (part of tiltingmodule 166, discussed below) configured to rotate a microfluidic device100 about one or more axes of rotation. In some embodiments, the tiltingdevice 190 is configured to support and/or hold the enclosure 102comprising the microfluidic circuit 120 about at least one axis suchthat the microfluidic device 100 (and thus the microfluidic circuit 120)can be held in a level orientation (i.e. at 0° relative to x- andy-axes), a vertical orientation (i.e. at 90° relative to the x-axisand/or the y-axis), or any orientation therebetween. The orientation ofthe microfluidic device 100 (and the microfluidic circuit 120) relativeto an axis is referred to herein as the “tilt” of the microfluidicdevice 100 (and the microfluidic circuit 120). For example, the tiltingdevice 190 can tilt the microfluidic device 100 at 0.1°, 0.2°, 0.3°,0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°,25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90° relativeto the x-axis or any degree therebetween. The level orientation (andthus the x- and y-axes) is defined as normal to a vertical axis definedby the force of gravity. The tilting device can also tilt themicrofluidic device 100 (and the microfluidic circuit 120) to any degreegreater than 90° relative to the x-axis and/or y-axis, or tilt themicrofluidic device 100 (and the microfluidic circuit 120) 180° relativeto the x-axis or the y-axis in order to fully invert the microfluidicdevice 100 (and the microfluidic circuit 120). Similarly, in someembodiments, the tilting device 190 tilts the microfluidic device 100(and the microfluidic circuit 120) about an axis of rotation defined byflow path 106 or some other portion of microfluidic circuit 120.

In some instances, the microfluidic device 100 is tilted into a verticalorientation such that the flow path 106 is positioned above or below oneor more sequestration pens. The term “above” as used herein denotes thatthe flow path 106 is positioned higher than the one or moresequestration pens on a vertical axis defined by the force of gravity(i.e. an object in a sequestration pen above a flow path 106 would havea higher gravitational potential energy than an object in the flowpath). The term “below” as used herein denotes that the flow path 106 ispositioned lower than the one or more sequestration pens on a verticalaxis defined by the force of gravity (i.e. an object in a sequestrationpen below a flow path 106 would have a lower gravitational potentialenergy than an object in the flow path).

In some instances, the tilting device 190 tilts the microfluidic device100 about an axis that is parallel to the flow path 106. Moreover, themicrofluidic device 100 can be tilted to an angle of less than 90° suchthat the flow path 106 is located above or below one or moresequestration pens without being located directly above or below thesequestration pens. In other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis perpendicular to the flow path106. In still other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis that is neither parallel norperpendicular to the flow path 106.

System 150 can further include a media source 178. The media source 178(e.g., a container, reservoir, or the like) can comprise multiplesections or containers, each for holding a different fluidic medium 180.Thus, the media source 178 can be a device that is outside of andseparate from the microfluidic device 100, as illustrated in FIG. 1A.Alternatively, the media source 178 can be located in whole or in partinside the enclosure 102 of the microfluidic device 100. For example,the media source 178 can comprise reservoirs that are part of themicrofluidic device 100.

FIG. 1A also illustrates simplified block diagram depictions of examplesof control and monitoring equipment 152 that constitute part of system150 and can be utilized in conjunction with a microfluidic device 100.As shown, examples of such control and monitoring equipment 152 includea master controller 154 comprising a media module 160 for controllingthe media source 178, a motive module 162 for controlling movementand/or selection of micro-objects (not shown) and/or medium (e.g.,droplets of medium) in the microfluidic circuit 120, an imaging module164 for controlling an imaging device 194 (e.g., a camera, microscope,light source or any combination thereof) for capturing images (e.g.,digital images), and a tilting module 166 for controlling a tiltingdevice 190. The control equipment 152 can also include other modules 168for controlling, monitoring, or performing other functions with respectto the microfluidic device 100. As shown, the equipment 152 can furtherinclude a display device 170 and an input/output device 172.

The master controller 154 can include a control module 156 and a digitalmemory 158. The control module 156 can comprise, for example, a digitalprocessor configured to operate in accordance with machine executableinstructions (e.g., software, firmware, source code, or the like) storedas non-transitory data or signals in the memory 158. Alternatively, orin addition, the control module 156 can comprise hardwired digitalcircuitry and/or analog circuitry. The media module 160, motive module162, imaging module 164, tilting module 166, and/or other modules 168can be similarly configured. Thus, functions, processes acts, actions,or steps of a process discussed herein as being performed with respectto the microfluidic device 100 or any other microfluidic apparatus canbe performed by any one or more of the master controller 154, mediamodule 160, motive module 162, imaging module 164, tilting module 166,and/or other modules 168 configured as discussed above. Similarly, themaster controller 154, media module 160, motive module 162, imagingmodule 164, tilting module 166, and/or other modules 168 may becommunicatively coupled to transmit and receive data used in anyfunction, process, act, action or step discussed herein.

The media module 160 controls the media source 178. For example, themedia module 160 can control the media source 178 to input a selectedfluidic medium 180 into the enclosure 102 (e.g., through an inlet port107). The media module 160 can also control removal of media from theenclosure 102 (e.g., through an outlet port (not shown)). One or moremedia can thus be selectively input into and removed from themicrofluidic circuit 120. The media module 160 can also control the flowof fluidic medium 180 in the flow path 106 inside the microfluidiccircuit 120. For example, in some embodiments media module 160 stops theflow of media 180 in the flow path 106 and through the enclosure 102prior to the tilting module 166 causing the tilting device 190 to tiltthe microfluidic device 100 to a desired angle of incline.

The motive module 162 can be configured to control selection, trapping,and movement of micro-objects (not shown) in the microfluidic circuit120. As discussed below with respect to FIGS. 1B and 1C, the enclosure102 can include a dielectrophoresis (DEP), optoelectronic tweezers (OET)and/or opto-electrowetting (OEW) configuration (not shown in FIG. 1A),and the motive module 162 can control the activation of electrodesand/or transistors (e.g., phototransistors) to select and movemicro-objects (not shown) and/or droplets of medium (not shown) in theflow path 106 and/or sequestration pens 124, 126, 128, 130.

The imaging module 164 can control the imaging device 194. For example,the imaging module 164 can receive and process image data from theimaging device 194. Image data from the imaging device 194 can compriseany type of information captured by the imaging device 194 (e.g., thepresence or absence of micro-objects, droplets of medium, accumulationof label, such as fluorescent label, etc.). Using the informationcaptured by the imaging device 194, the imaging module 164 can furthercalculate the position of objects (e.g., micro-objects, droplets ofmedium) and/or the rate of motion of such objects within themicrofluidic device 100.

The tilting module 166 can control the tilting motions of tilting device190. Alternatively, or in addition, the tilting module 166 can controlthe tilting rate and timing to optimize transfer of micro-objects to theone or more sequestration pens via gravitational forces. The tiltingmodule 166 is communicatively coupled with the imaging module 164 toreceive data describing the motion of micro-objects and/or droplets ofmedium in the microfluidic circuit 120. Using this data, the tiltingmodule 166 may adjust the tilt of the microfluidic circuit 120 in orderto adjust the rate at which micro-objects and/or droplets of medium movein the microfluidic circuit 120. The tilting module 166 may also usethis data to iteratively adjust the position of a micro-object and/ordroplet of medium in the microfluidic circuit 120.

In the example shown in FIG. 1A, the microfluidic circuit 120 isillustrated as including a microfluidic channel 122 and sequestrationpens 124, 126, 128, 130. Each pen includes an opening to channel 122,but otherwise is enclosed such that the pens can substantially isolatemicro-objects inside the pen from fluidic medium 180 and/ormicro-objects in the flow path 106 of channel 122 or in other pens. Thewalls of the sequestration pen extend from the inner surface 109 of thebase to the inside surface of the cover 110 to provide enclosure. Theopening of the pen to the microfluidic channel 122 is oriented at anangle to the flow 106 of fluidic medium 180 such that flow 106 is notdirected into the pens. The flow may be tangential or orthogonal to theplane of the opening of the pen. In some instances, pens 124, 126, 128,130 are configured to physically corral one or more micro-objects withinthe microfluidic circuit 120. Sequestration pens in accordance with thepresent disclosure can comprise various shapes, surfaces and featuresthat are optimized for use with DEP, OET, OEW, fluid flow, and/orgravitational forces, as will be discussed and shown in detail below.

The microfluidic circuit 120 may include any number of microfluidicsequestration pens. Although five sequestration pens are shown,microfluidic circuit 120 may have fewer or more sequestration pens. Asshown, microfluidic sequestration pens 124, 126, 128, and 130 ofmicrofluidic circuit 120 each comprise differing features and shapeswhich may provide one or more benefits useful in producing an embryo,such as isolating one ovum from an adjacent ovum. Testing, stimulatingand fertilizing may all be performed on an individual basis and, in someembodiments, may be performed on an individual time scale. In someembodiments, the microfluidic circuit 120 comprises a plurality ofidentical microfluidic sequestration pens.

In some embodiments, the microfluidic circuit 120 comprises a pluralityof microfluidic sequestration pens, wherein two or more of thesequestration pens comprise differing structures and/or features whichprovide differing benefits in producing embryos. One non-limitingexample may include maintaining ova in one type of pen while maintainingsperm in a different type of pen. In another embodiment, at least one ofthe sequestration pens is configured to have electrical contactssuitable for providing electrical activation for an ovum. In yet anotherembodiment, differing types of cells (such as, for example, uterinecells, endometrial cells, PEG (intercalary) cells derived from theuterine tube (e.g., oviduct or Fallopian tube), cumulus cells, or acombination thereof) may be disposed in sequestration pens adjacent to asequestration pen containing an ovum, such that secretions from thesurrounding sequestration pens may diffuse out of each respective penand into the pen containing an ovum, which is not possible withmacroscale in-vitro culturing and fertilization. Microfluidic devicesuseful for producing an embryo may include any of the sequestration pens124, 126, 128, and 130 or variations thereof, and/or may include pensconfigured like those shown in FIGS. 2B, 2C, 2D, 2E and 2F, as discussedbelow.

In the embodiment illustrated in FIG. 1A, a single channel 122 and flowpath 106 is shown. However, other embodiments may contain multiplechannels 122, each configured to include a flow path 106. Themicrofluidic circuit 120 further includes an inlet valve or port 107 influid communication with the flow path 106 and fluidic medium 180,whereby fluidic medium 180 can access channel 122 via the inlet port107. In some instances, the flow path 106 comprises a single path. Insome instances, the single path is arranged in a zigzag pattern wherebythe flow path 106 travels across the microfluidic device 100 two or moretimes in alternating directions.

In some instances, microfluidic circuit 120 includes a plurality ofparallel channels 122 and flow paths 106, wherein the fluidic medium 180within each flow path 106 flows in the same direction. In someinstances, the fluidic medium within each flow path 106 flows in atleast one of a forward or reverse direction. In some instances, aplurality of sequestration pens is configured (e.g., relative to achannel 122) such that the sequestration pens can be loaded with targetmicro-objects in parallel.

In some embodiments, microfluidic circuit 120 further includes one ormore micro-object traps 132. The traps 132 are generally formed in awall forming the boundary of a channel 122, and may be positionedopposite an opening of one or more of the microfluidic sequestrationpens 124, 126, 128, 130. In some embodiments, the traps 132 areconfigured to receive or capture a single micro-object from the flowpath 106. In some embodiments, the traps 132 are configured to receiveor capture a plurality of micro-objects from the flow path 106. In someinstances, the traps 132 comprise a volume approximately equal to thevolume of a single target micro-object.

The traps 132 may further include an opening which is configured toassist the flow of targeted micro-objects into the traps 132. In someinstances, the traps 132 include an opening having a height and widththat is approximately equal to the dimensions of a single targetmicro-object, whereby larger micro-objects are prevented from enteringinto the micro-object trap. The traps 132 may further include otherfeatures configured to assist in retention of targeted micro-objectswithin the trap 132. In some instances, the trap 132 is aligned with andsituated on the opposite side of a channel 122 relative to the openingof a microfluidic sequestration pen, such that upon tilting themicrofluidic device 100 about an axis parallel to the microfluidicchannel 122, the trapped micro-object exits the trap 132 at a trajectorythat causes the micro-object to fall into the opening of thesequestration pen. In some instances, the trap 132 includes a sidepassage 134 that is smaller than the target micro-object in order tofacilitate flow through the trap 132 and thereby increase the likelihoodof capturing a micro-object in the trap 132.

In some embodiments, dielectrophoretic (DEP) forces are applied acrossthe fluidic medium 180 (e.g., in the flow path and/or in thesequestration pens) via one or more electrodes (not shown) tomanipulate, transport, separate and sort micro-objects located therein.For example, in some embodiments, DEP forces are applied to one or moreportions of microfluidic circuit 120 in order to transfer a singlemicro-object from the flow path 106 into a desired microfluidicsequestration pen. In some embodiments, DEP forces are used to prevent amicro-object within a sequestration pen (e.g., sequestration pen 124,126, 128, or 130) from being displaced therefrom. Further, in someembodiments, DEP forces are used to selectively remove a micro-objectfrom a sequestration pen that was previously collected in accordancewith the embodiments of the current disclosure. In some embodiments, theDEP forces comprise optoelectronic tweezer (OET) forces.

In other embodiments, optoelectrowetting (OEW) forces are applied to oneor more positions in the support structure 104 (and/or the cover 110) ofthe microfluidic device 100 (e.g., positions helping to define the flowpath and/or the sequestration pens) via one or more electrodes (notshown) to manipulate, transport, separate and sort droplets located inthe microfluidic circuit 120. For example, in some embodiments, OEWforces are applied to one or more positions in the support structure 104(and/or the cover 110) in order to transfer a single droplet from theflow path 106 into a desired microfluidic sequestration pen. In someembodiments, OEW forces are used to prevent a droplet within asequestration pen (e.g., sequestration pen 124, 126, 128, or 130) frombeing displaced therefrom. Further, in some embodiments, OEW forces areused to selectively remove a droplet from a sequestration pen that waspreviously collected in accordance with the embodiments of the currentdisclosure.

In some embodiments, DEP and/or OEW forces are combined with otherforces, such as flow and/or gravitational force, so as to manipulate,transport, separate and sort micro-objects and/or droplets within themicrofluidic circuit 120. For example, the enclosure 102 can be tilted(e.g., by tilting device 190) to position the flow path 106 andmicro-objects located therein above the microfluidic sequestration pens,and the force of gravity can transport the micro-objects and/or dropletsinto the pens. In some embodiments, the DEP and/or OEW forces can beapplied prior to the other forces. In other embodiments, the DEP and/orOEW forces can be applied after the other forces. In still otherinstances, the DEP and/or OEW forces can be applied at the same time asthe other forces or in an alternating manner with the other forces.

FIGS. 1B, 1C, and 2A-2H illustrates various embodiments of microfluidicdevices that can be used in the practice of the embodiments of thepresent disclosure. FIG. 1B depicts an embodiment in which themicrofluidic device 200 is configured as an optically-actuatedelectrokinetic device. A variety of optically-actuated electrokineticdevices are known in the art, including devices having an optoelectronictweezer (OET) configuration and devices having an opto-electrowetting(OEW) configuration. Examples of suitable OET configurations areillustrated in the following U.S. patent documents, each of which isincorporated herein by reference in its entirety: U.S. Pat. No. RE44,711 (Wu et al.) (originally issued as U.S. Pat. No. 7,612,355); andU.S. Pat. No. 7,956,339 (Ohta et al.). Examples of OEW configurationsare illustrated in U.S. Pat. No. 6,958,132 (Chiou et al.) and U.S.Patent Application Publication No. 2012/0024708 (Chiou et al.), both ofwhich are incorporated by reference herein in their entirety. Yetanother example of an optically-actuated electrokinetic device includesa combined OET/OEW configuration, examples of which are shown in U.S.Patent Publication Nos. 20150306598 (Khandros et al.) and 20150306599(Khandros et al.) and their corresponding PCT Publications WO2015/164846and WO2015/164847, all of which are incorporated herein by reference intheir entirety.

Examples of microfluidic devices having pens in which oocytes, ova, orembryos can be placed, cultured, and/or monitored have been described,for example, in US 2014/0116881 (application Ser. No. 14/060,117, filedOct. 22, 2013), US 2015/0151298 (application Ser. No. 14/520,568, filedOct. 22, 2014), and US 2015/0165436 (application Ser. No. 14/521,447,filed Oct. 22, 2014), each of which is incorporated herein by referencein its entirety. U.S. application Ser. Nos. 14/520,568 and 14/521,447also describe exemplary methods of analyzing secretions of cellscultured in a microfluidic device. Each of the foregoing applicationsfurther describes microfluidic devices configured to producedielectrophoretic (DEP) forces, such as optoelectronic tweezers (OET) orconfigured to provide opto-electro wetting (OEW). For example, theoptoelectronic tweezers device illustrated in FIG. 2 of US 2014/0116881is an example of a device that can be utilized in embodiments of thepresent disclosure to select and move an individual biologicalmicro-object or a group of biological micro-objects.

Microfluidic device motive configurations. As described above, thecontrol and monitoring equipment of the system can include a motivemodule for selecting and moving objects, such as micro-objects ordroplets, in the microfluidic circuit of a microfluidic device. Themicrofluidic device can have a variety of motive configurations,depending upon the type of object being moved and other considerations.For example, a dielectrophoresis (DEP) configuration can be utilized toselect and move micro-objects in the microfluidic circuit. Thus, thesupport structure 104 and/or cover 110 of the microfluidic device 100can comprise a DEP configuration for selectively inducing DEP forces onmicro-objects in a fluidic medium 180 in the microfluidic circuit 120and thereby select, capture, and/or move individual micro-objects orgroups of micro-objects. Alternatively, the support structure 104 and/orcover 110 of the microfluidic device 100 can comprise an electrowetting(EW) configuration for selectively inducing EW forces on droplets in afluidic medium 180 in the microfluidic circuit 120 and thereby select,capture, and/or move individual droplets or groups of droplets.

One example of a microfluidic device 200 comprising a DEP configurationis illustrated in FIGS. 1B and 1C. While for purposes of simplicityFIGS. 1B and 1C show a side cross-sectional view and a topcross-sectional view, respectively, of a portion of an enclosure 102 ofthe microfluidic device 200 having an open region/chamber 202, it shouldbe understood that the region/chamber 202 may be part of a fluidiccircuit element having a more detailed structure, such as a growthchamber, a sequestration pen, a flow region, or a flow channel.Furthermore, the microfluidic device 200 may include other fluidiccircuit elements. For example, the microfluidic device 200 can include aplurality of growth chambers or sequestration pens and/or one or moreflow regions or flow channels, such as those described herein withrespect to microfluidic device 100. A DEP configuration may beincorporated into any such fluidic circuit elements of the microfluidicdevice 200, or select portions thereof. It should be further appreciatedthat any of the above or below described microfluidic device componentsand system components may be incorporated in and/or used in combinationwith the microfluidic device 200. For example, system 150 includingcontrol and monitoring equipment 152, described above, may be used withmicrofluidic device 200, including one or more of the media module 160,motive module 162, imaging module 164, tilting module 166, and othermodules 168.

As seen in FIG. 1B, the microfluidic device 200 includes a supportstructure 104 having a bottom electrode 204 and an electrode activationsubstrate 206 overlying the bottom electrode 204, and a cover 110 havinga top electrode 210, with the top electrode 210 spaced apart from thebottom electrode 204. The top electrode 210 and the electrode activationsubstrate 206 define opposing surfaces of the region/chamber 202. Amedium 180 contained in the region/chamber 202 thus provides a resistiveconnection between the top electrode 210 and the electrode activationsubstrate 206. A power source 212 configured to be connected to thebottom electrode 204 and the top electrode 210 and create a biasingvoltage between the electrodes, as required for the generation of DEPforces in the region/chamber 202, is also shown. The power source 212can be, for example, an alternating current (AC) power source.

In certain embodiments, the microfluidic device 200 illustrated in FIGS.1B and 1C can have an optically-actuated DEP configuration. Accordingly,changing patterns of light 218 from the light source 216, which may becontrolled by the motive module 162, can selectively activate anddeactivate changing patterns of DEP electrodes at regions 214 of theinner surface 208 of the electrode activation substrate 206.(Hereinafter the regions 214 of a microfluidic device having a DEPconfiguration are referred to as “DEP electrode regions.”) Asillustrated in FIG. 1C, a light pattern 218 directed onto the innersurface 208 of the electrode activation substrate 206 can illuminateselect DEP electrode regions 214 a (shown in white) in a pattern, suchas a square. The non-illuminated DEP electrode regions 214(cross-hatched) are hereinafter referred to as “dark” DEP electroderegions 214. The relative electrical impedance through the DEP electrodeactivation substrate 206 (i.e., from the bottom electrode 204 up to theinner surface 208 of the electrode activation substrate 206 whichinterfaces with the medium 180 in the flow region 106) is greater thanthe relative electrical impedance through the medium 180 in theregion/chamber 202 (i.e., from the inner surface 208 of the electrodeactivation substrate 206 to the top electrode 210 of the cover 110) ateach dark DEP electrode region 214. An illuminated DEP electrode region214 a, however, exhibits a reduced relative impedance through theelectrode activation substrate 206 that is less than the relativeimpedance through the medium 180 in the region/chamber 202 at eachilluminated DEP electrode region 214 a.

With the power source 212 activated, the foregoing DEP configurationcreates an electric field gradient in the fluidic medium 180 betweenilluminated DEP electrode regions 214 a and adjacent dark DEP electroderegions 214, which in turn creates local DEP forces that attract orrepel nearby micro-objects (not shown) in the fluidic medium 180. DEPelectrodes that attract or repel micro-objects in the fluidic medium 180can thus be selectively activated and deactivated at many different suchDEP electrode regions 214 at the inner surface 208 of the region/chamber202 by changing light patterns 218 projected from a light source 216into the microfluidic device 200. Whether the DEP forces attract orrepel nearby micro-objects can depend on such parameters as thefrequency of the power source 212 and the dielectric properties of themedium 180 and/or micro-objects (not shown).

The square pattern 220 of illuminated DEP electrode regions 214 aillustrated in FIG. 1C is an example only. Any pattern of the DEPelectrode regions 214 can be illuminated (and thereby activated) by thepattern of light 218 projected into the microfluidic device 200, and thepattern of illuminated/activated DEP electrode regions 214 can berepeatedly changed by changing or moving the light pattern 218.

In some embodiments, the electrode activation substrate 206 can compriseor consist of a photoconductive material. In such embodiments, the innersurface 208 of the electrode activation substrate 206 can befeatureless. For example, the electrode activation substrate 206 cancomprise or consist of a layer of hydrogenated amorphous silicon(a-Si:H). The a-Si:H can comprise, for example, about 8% to 40% hydrogen(calculated as 100*the number of hydrogen atoms/the total number ofhydrogen and silicon atoms). The layer of a-Si:H can have a thickness ofabout 500 nm to about 2.0 μm. In such embodiments, the DEP electroderegions 214 can be created anywhere and in any pattern on the innersurface 208 of the electrode activation substrate 206, in accordancewith the light pattern 218. The number and pattern of the DEP electroderegions 214 thus need not be fixed, but can correspond to the lightpattern 218. Examples of microfluidic devices having a DEP configurationcomprising a photoconductive layer such as discussed above have beendescribed, for example, in U.S. Pat. No. RE 44,711 (Wu et al.)(originally issued as U.S. Pat. No. 7,612,355), the entire contents ofwhich are incorporated herein by reference.

In other embodiments, the electrode activation substrate 206 cancomprise a substrate comprising a plurality of doped layers,electrically insulating layers (or regions), and electrically conductivelayers that form semiconductor integrated circuits, such as is known insemiconductor fields. For example, the electrode activation substrate206 can comprise a plurality of phototransistors, including, forexample, lateral bipolar phototransistors, each phototransistorcorresponding to a DEP electrode region 214. Alternatively, theelectrode activation substrate 206 can comprise electrodes (e.g.,conductive metal electrodes) controlled by phototransistor switches,with each such electrode corresponding to a DEP electrode region 214.The electrode activation substrate 206 can include a pattern of suchphototransistors or phototransistor-controlled electrodes. The pattern,for example, can be an array of substantially square phototransistors orphototransistor-controlled electrodes arranged in rows and columns, suchas shown in FIG. 2B. Alternatively, the pattern can be an array ofsubstantially hexagonal phototransistors or phototransistor-controlledelectrodes that form a hexagonal lattice. Regardless of the pattern,electric circuit elements can form electrical connections between theDEP electrode regions 214 at the inner surface 208 of the electrodeactivation substrate 206 and the bottom electrode 204, and thoseelectrical connections (i.e., phototransistors or electrodes) can beselectively activated and deactivated by the light pattern 218. When notactivated, each electrical connection can have high impedance such thatthe relative impedance through the electrode activation substrate 206(i.e., from the bottom electrode 204 to the inner surface 208 of theelectrode activation substrate 206 which interfaces with the medium 180in the region/chamber 202) is greater than the relative impedancethrough the medium 180 (i.e., from the inner surface 208 of theelectrode activation substrate 206 to the top electrode 210 of the cover110) at the corresponding DEP electrode region 214. When activated bylight in the light pattern 218, however, the relative impedance throughthe electrode activation substrate 206 is less than the relativeimpedance through the medium 180 at each illuminated DEP electroderegion 214, thereby activating the DEP electrode at the correspondingDEP electrode region 214 as discussed above. DEP electrodes that attractor repel micro-objects (not shown) in the medium 180 can thus beselectively activated and deactivated at many different DEP electroderegions 214 at the inner surface 208 of the electrode activationsubstrate 206 in the region/chamber 202 in a manner determined by thelight pattern 218.

Examples of microfluidic devices having electrode activation substratesthat comprise phototransistors have been described, for example, in U.S.Pat. No. 7,956,339 (Ohta et al.) (see, e.g., device 300 illustrated inFIGS. 21 and 22 , and descriptions thereof), the entire contents ofwhich are incorporated herein by reference. Examples of microfluidicdevices having electrode activation substrates that comprise electrodescontrolled by phototransistor switches have been described, for example,in U.S. Patent Publication No. 2014/0124370 (Short et al.) (see, e.g.,devices 200, 400, 500, 600, and 900 illustrated throughout the drawings,and descriptions thereof), the entire contents of which are incorporatedherein by reference.

In some embodiments of a DEP configured microfluidic device, the topelectrode 210 is part of a first wall (or cover 110) of the enclosure102, and the electrode activation substrate 206 and bottom electrode 204are part of a second wall (or support structure 104) of the enclosure102. The region/chamber 202 can be between the first wall and the secondwall. In other embodiments, the electrode 210 is part of the second wall(or support structure 104) and one or both of the electrode activationsubstrate 206 and/or the electrode 210 are part of the first wall (orcover 110). Moreover, the light source 216 can alternatively be used toilluminate the enclosure 102 from below.

With the microfluidic device 200 of FIGS. 1B-1C having a DEPconfiguration, the motive module 162 can select a micro-object (notshown) in the medium 180 in the region/chamber 202 by projecting a lightpattern 218 into the microfluidic device 200 to activate a first set ofone or more DEP electrodes at DEP electrode regions 214 a of the innersurface 208 of the electrode activation substrate 206 in a pattern(e.g., square pattern 220) that surrounds and captures the micro-object.The motive module 162 can then move the in situ-generated capturedmicro-object by moving the light pattern 218 relative to themicrofluidic device 200 to activate a second set of one or more DEPelectrodes at DEP electrode regions 214. Alternatively, the microfluidicdevice 200 can be moved relative to the light pattern 218.

In other embodiments, the microfluidic device 200 can have a DEPconfiguration that does not rely upon light activation of DEP electrodesat the inner surface 208 of the electrode activation substrate 206. Forexample, the electrode activation substrate 206 can comprise selectivelyaddressable and energizable electrodes positioned opposite to a surfaceincluding at least one electrode (e.g., cover 110). Switches (e.g.,transistor switches in a semiconductor substrate) may be selectivelyopened and closed to activate or inactivate DEP electrodes at DEPelectrode regions 214, thereby creating a net DEP force on amicro-object (not shown) in region/chamber 202 in the vicinity of theactivated DEP electrodes. Depending on such characteristics as thefrequency of the power source 212 and the dielectric properties of themedium (not shown) and/or micro-objects in the region/chamber 202, theDEP force can attract or repel a nearby micro-object. By selectivelyactivating and deactivating a set of DEP electrodes (e.g., at a set ofDEP electrodes regions 214 that forms a square pattern 220), one or moremicro-objects in region/chamber 202 can be trapped and moved within theregion/chamber 202. The motive module 162 in FIG. 1A can control suchswitches and thus activate and deactivate individual ones of the DEPelectrodes to select, trap, and move particular micro-objects (notshown) around the region/chamber 202. Microfluidic devices having a DEPconfiguration that includes selectively addressable and energizableelectrodes are known in the art and have been described, for example, inU.S. Pat. No. 6,294,063 (Becker et al.) and U.S. Pat. No. 6,942,776(Medoro), the entire contents of which are incorporated herein byreference.

As yet another example, the microfluidic device 200 can have anelectrowetting (EW) configuration, which can be in place of the DEPconfiguration or can be located in a portion of the microfluidic device200 that is separate from the portion which has the DEP configuration.The EW configuration can be an opto-electrowetting configuration or anelectrowetting on dielectric (EWOD) configuration, both of which areknown in the art. In some EW configurations, the support structure 104has an electrode activation substrate 206 sandwiched between adielectric layer (not shown) and the bottom electrode 204. Thedielectric layer can comprise a hydrophobic material and/or can becoated with a hydrophobic material, as described below. For microfluidicdevices 200 that have an EW configuration, the inner surface 208 of thesupport structure 104 is the inner surface of the dielectric layer orits hydrophobic coating.

The dielectric layer (not shown) can comprise one or more oxide layers,and can have a thickness of about 50 nm to about 250 nm (e.g., about 125nm to about 175 nm). In certain embodiments, the dielectric layer maycomprise a layer of oxide, such as a metal oxide (e.g., aluminum oxideor hafnium oxide). In certain embodiments, the dielectric layer cancomprise a dielectric material other than a metal oxide, such as siliconoxide or a nitride. Regardless of the exact composition and thickness,the dielectric layer can have an impedance of about 10 kOhms to about 50kOhms.

In some embodiments, the surface of the dielectric layer that facesinward toward region/chamber 202 is coated with a hydrophobic material.The hydrophobic material can comprise, for example, fluorinated carbonmolecules. Examples of fluorinated carbon molecules includeperfluoro-polymers such as polytetrafluoroethylene (e.g., TEFLON®) orpoly(2,3-difluoromethylenyl-perfluorotetrahydrofuran) (e.g., CYTOP™).Molecules that make up the hydrophobic material can be covalently bondedto the surface of the dielectric layer. For example, molecules of thehydrophobic material can be covalently bound to the surface of thedielectric layer by means of a linker such as a siloxane group, aphosphonic acid group, or a thiol group. Thus, in some embodiments, thehydrophobic material can comprise alkyl-terminated siloxane,alkyl-termination phosphonic acid, or alkyl-terminated thiol. The alkylgroup can be long-chain hydrocarbons (e.g., having a chain of at least10 carbons, or at least 16, 18, 20, 22, or more carbons). Alternatively,fluorinated (or perfluorinated) carbon chains can be used in place ofthe alkyl groups. Thus, for example, the hydrophobic material cancomprise fluoroalkyl-terminated siloxane, fluoroalkyl-terminatedphosphonic acid, or fluoroalkyl-terminated thiol. In some embodiments,the hydrophobic coating has a thickness of about 10 nm to about 50 nm.In other embodiments, the hydrophobic coating has a thickness of lessthan 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm).

In some embodiments, the cover 110 of a microfluidic device 200 havingan electrowetting configuration is coated with a hydrophobic material(not shown) as well. The hydrophobic material can be the samehydrophobic material used to coat the dielectric layer of the supportstructure 104, and the hydrophobic coating can have a thickness that issubstantially the same as the thickness of the hydrophobic coating onthe dielectric layer of the support structure 104. Moreover, the cover110 can comprise an electrode activation substrate 206 sandwichedbetween a dielectric layer and the top electrode 210, in the manner ofthe support structure 104. The electrode activation substrate 206 andthe dielectric layer of the cover 110 can have the same compositionand/or dimensions as the electrode activation substrate 206 and thedielectric layer of the support structure 104. Thus, the microfluidicdevice 200 can have two electrowetting surfaces.

In some embodiments, the electrode activation substrate 206 can comprisea photoconductive material, such as described above. Accordingly, incertain embodiments, the electrode activation substrate 206 can compriseor consist of a layer of hydrogenated amorphous silicon (a-Si:H). Thea-Si:H can comprise, for example, about 8% to 40% hydrogen (calculatedas 100*the number of hydrogen atoms/the total number of hydrogen andsilicon atoms). The layer of a-Si:H can have a thickness of about 500 nmto about 2.0 μm. Alternatively, the electrode activation substrate 206can comprise electrodes (e.g., conductive metal electrodes) controlledby phototransistor switches, as described above. Microfluidic deviceshaving an opto-electrowetting configuration are known in the art and/orcan be constructed with electrode activation substrates known in theart. For example, U.S. Pat. No. 6,958,132 (Chiou et al.), the entirecontents of which are incorporated herein by reference, disclosesopto-electrowetting configurations having a photoconductive materialsuch as a-Si:H, while U.S. Patent Publication No. 2014/0124370 (Short etal.), referenced above, discloses electrode activation substrates havingelectrodes controlled by phototransistor switches.

The microfluidic device 200 thus can have an opto-electrowettingconfiguration, and light patterns 218 can be used to activatephotoconductive EW regions or photoresponsive EW electrodes in theelectrode activation substrate 206. Such activated EW regions or EWelectrodes of the electrode activation substrate 206 can generate anelectrowetting force at the inner surface 208 of the support structure104 (i.e., the inner surface of the overlaying dielectric layer or itshydrophobic coating). By changing the light patterns 218 (or movingmicrofluidic device 200 relative to the light source 216) incident onthe electrode activation substrate 206, droplets (e.g., containing anaqueous medium, solution, or solvent) contacting the inner surface 208of the support structure 104 can be moved through an immiscible fluid(e.g., an oil medium) present in the region/chamber 202.

In other embodiments, microfluidic devices 200 can have an EWODconfiguration, and the electrode activation substrate 206 can compriseselectively addressable and energizable electrodes that do not rely uponlight for activation. The electrode activation substrate 206 thus caninclude a pattern of such electrowetting (EW) electrodes. The pattern,for example, can be an array of substantially square EW electrodesarranged in rows and columns, such as shown in FIG. 2B. Alternatively,the pattern can be an array of substantially hexagonal EW electrodesthat form a hexagonal lattice. Regardless of the pattern, the EWelectrodes can be selectively activated (or deactivated) by electricalswitches (e.g., transistor switches in a semiconductor substrate). Byselectively activating and deactivating EW electrodes in the electrodeactivation substrate 206, droplets (not shown) contacting the innersurface 208 of the overlaying dielectric layer or its hydrophobiccoating can be moved within the region/chamber 202. The motive module162 in FIG. 1A can control such switches and thus activate anddeactivate individual EW electrodes to select and move particulardroplets around region/chamber 202. Microfluidic devices having a EWODconfiguration with selectively addressable and energizable electrodesare known in the art and have been described, for example, in U.S. Pat.No. 8,685,344 (Sundarsan et al.), the entire contents of which areincorporated herein by reference.

Regardless of the configuration of the microfluidic device 200, a powersource 212 can be used to provide a potential (e.g., an AC voltagepotential) that powers the electrical circuits of the microfluidicdevice 200. The power source 212 can be the same as, or a component of,the power source 192 referenced in FIG. 1 . Power source 212 can beconfigured to provide an AC voltage and/or current to the top electrode210 and the bottom electrode 204. For an AC voltage, the power source212 can provide a frequency range and an average or peak power (e.g.,voltage or current) range sufficient to generate net DEP forces (orelectrowetting forces) strong enough to trap and move individualmicro-objects (not shown) in the region/chamber 202, as discussed above,and/or to change the wetting properties of the inner surface 208 of thesupport structure 104 (i.e., the dielectric layer and/or the hydrophobiccoating on the dielectric layer) in the region/chamber 202, as alsodiscussed above. Such frequency ranges and average or peak power rangesare known in the art. See, e.g., U.S. Pat. No. 6,958,132 (Chiou et al.),U.S. Pat. No. RE44,711 (Wu et al.) (originally issued as U.S. Pat. No.7,612,355), and US Patent Application Publication Nos. US2014/0124370(Short et al.), US2015/0306598 (Khandros et al.), and US2015/0306599(Khandros et al.).

Sequestration pens. Non-limiting examples of generic sequestration pens224, 226, and 228 are shown within the microfluidic device 230 depictedin FIGS. 2A-2C. Each sequestration pen 224, 226, and 228 can comprise anisolation structure 232 defining an isolation region 240 and aconnection region 236 fluidically connecting the isolation region 240 toa channel 122. The connection region 236 can include a proximal opening234 to the microfluidic channel 122 and a distal opening 238 to theisolation region 240. The connection region 236 can be configured sothat the maximum penetration depth of a flow of a fluidic medium (notshown) flowing from the microfluidic channel 122 into the sequestrationpen 224, 226, 228 does not extend into the isolation region 240. Thus,due to the connection region 236, a micro-object (not shown) or othermaterial (not shown) disposed in an isolation region 240 of asequestration pen 224, 226, 228 can thus be isolated from, and notsubstantially affected by, a flow of medium 180 in the microfluidicchannel 122.

The sequestration pens 224, 226, and 228 of FIGS. 2A-2C each have asingle opening which opens directly to the microfluidic channel 122. Theopening of the sequestration pen opens laterally from the microfluidicchannel 122. The electrode activation substrate 206 underlays both themicrofluidic channel 122 and the sequestration pens 224, 226, and 228.The upper surface of the electrode activation substrate 206 within theenclosure of a sequestration pen, forming the floor of the sequestrationpen, is disposed at the same level or substantially the same level ofthe upper surface the of electrode activation substrate 206 within themicrofluidic channel 122 (or flow region if a channel is not present),forming the floor of the flow channel (or flow region, respectively) ofthe microfluidic device. The electrode activation substrate 206 may befeatureless or may have an irregular or patterned surface that variesfrom its highest elevation to its lowest depression by less than about 3microns, 2.5 microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.5microns, 0.4 microns, 0.2 microns, 0.1 microns or less. The variation ofelevation in the upper surface of the substrate across both themicrofluidic channel 122 (or flow region) and sequestration pens may beless than about 3%, 2%, 1%. 0.9%, 0.8%, 0.5%, 0.3% or 0.1% of the heightof the walls of the sequestration pen or walls of the microfluidicdevice. While described in detail for the microfluidic device 200, thisalso applies to any of the microfluidic devices 100, 230, 250, 280, 290,320, 400, 450, 500, 700 described herein.

The microfluidic channel 122 can thus be an example of a swept region,and the isolation regions 240 of the sequestration pens 224, 226, 228can be examples of unswept regions. As noted, the microfluidic channel122 and sequestration pens 224, 226, 228 can be configured to containone or more fluidic media 180. In the example shown in FIGS. 2A-2B, theports 222 are connected to the microfluidic channel 122 and allow afluidic medium 180 to be introduced into or removed from themicrofluidic device 230. Prior to introduction of the fluidic medium180, the microfluidic device may be primed with a gas such as carbondioxide gas. Once the microfluidic device 230 contains the fluidicmedium 180, the flow 242 of fluidic medium 180 in the microfluidicchannel 122 can be selectively generated and stopped. For example, asshown, the ports 222 can be disposed at different locations (e.g.,opposite ends) of the microfluidic channel 122, and a flow 242 of mediumcan be created from one port 222 functioning as an inlet to another port222 functioning as an outlet.

FIG. 2C illustrates a detailed view of an example of a sequestration pen224 according to the present disclosure. Examples of micro-objects 246are also shown.

As is known, a flow 242 of fluidic medium 180 in a microfluidic channel122 past a proximal opening 234 of sequestration pen 224 can cause asecondary flow 244 of the medium 180 into and/or out of thesequestration pen 224. To isolate micro-objects 246 in the isolationregion 240 of a sequestration pen 224 from the secondary flow 244, thelength L_(con) of the connection region 236 of the sequestration pen 224(i.e., from the proximal opening 234 to the distal opening 238) shouldbe greater than the penetration depth D_(p) of the secondary flow 244into the connection region 236. The penetration depth D_(p) of thesecondary flow 244 depends upon the velocity of the fluidic medium 180flowing in the microfluidic channel 122 and various parameters relatingto the configuration of the microfluidic channel 122 and the proximalopening 234 of the connection region 236 to the microfluidic channel122. For a given microfluidic device, the configurations of themicrofluidic channel 122 and the opening 234 will be fixed, whereas therate of flow 242 of fluidic medium 180 in the microfluidic channel 122will be variable. Accordingly, for each sequestration pen 224, a maximalvelocity V_(max) for the flow 242 of fluidic medium 180 in channel 122can be identified that ensures that the penetration depth D_(p) of thesecondary flow 244 does not exceed the length L_(con) of the connectionregion 236. As long as the rate of the flow 242 of fluidic medium 180 inthe microfluidic channel 122 does not exceed the maximum velocityV_(max), the resulting secondary flow 244 can be limited to themicrofluidic channel 122 and the connection region 236 and kept out ofthe isolation region 240. The flow 242 of medium 180 in the microfluidicchannel 122 will thus not draw micro-objects 246 out of the isolationregion 240. Rather, micro-objects 246 located in the isolation region240 will stay in the isolation region 240 regardless of the flow 242 offluidic medium 180 in the microfluidic channel 122.

Moreover, as long as the rate of flow 242 of medium 180 in themicrofluidic channel 122 does not exceed V_(max), the flow 242 offluidic medium 180 in the microfluidic channel 122 will not movemiscellaneous particles (e.g., microparticles and/or nanoparticles) fromthe microfluidic channel 122 into the isolation region 240 of asequestration pen 224. Having the length L_(con) of the connectionregion 236 be greater than the maximum penetration depth D_(p) of thesecondary flow 244 can thus prevent contamination of one sequestrationpen 224 with miscellaneous particles from the microfluidic channel 122or another sequestration pen (e.g., sequestration pens 226, 228 in FIG.2D).

Because the microfluidic channel 122 and the connection regions 236 ofthe sequestration pens 224, 226, 228 can be affected by the flow 242 ofmedium 180 in the microfluidic channel 122, the microfluidic channel 122and connection regions 236 can be deemed swept (or flow) regions of themicrofluidic device 230. The isolation regions 240 of the sequestrationpens 224, 226, 228, on the other hand, can be deemed unswept (ornon-flow) regions. For example, components (not shown) in a firstfluidic medium 180 in the microfluidic channel 122 can mix with a secondfluidic medium 248 in the isolation region 240 substantially only bydiffusion of components of the first medium 180 from the microfluidicchannel 122 through the connection region 236 and into the secondfluidic medium 248 in the isolation region 240. Similarly, components(not shown) of the second medium 248 in the isolation region 240 can mixwith the first medium 180 in the microfluidic channel 122 substantiallyonly by diffusion of components of the second medium 248 from theisolation region 240 through the connection region 236 and into thefirst medium 180 in the microfluidic channel 122. In some embodiments,the extent of fluidic medium exchange between the isolation region of asequestration pen and the flow region by diffusion is greater than about90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or greater than about 99% offluidic exchange. The first medium 180 can be the same medium or adifferent medium than the second medium 248. Moreover, the first medium180 and the second medium 248 can start out being the same, then becomedifferent (e.g., through conditioning of the second medium 248 by one ormore cells in the isolation region 240, or by changing the medium 180flowing through the microfluidic channel 122).

The maximum penetration depth D_(p) of the secondary flow 244 caused bythe flow 242 of fluidic medium 180 in the microfluidic channel 122 candepend on a number of parameters, as mentioned above. Examples of suchparameters include: the shape of the microfluidic channel 122 (e.g., themicrofluidic channel can direct medium into the connection region 236,divert medium away from the connection region 236, or direct medium in adirection substantially perpendicular to the proximal opening 234 of theconnection region 236 to the microfluidic channel 122); a width W_(ch)(or cross-sectional area) of the microfluidic channel 122 at theproximal opening 234; and a width W_(con) (or cross-sectional area) ofthe connection region 236 at the proximal opening 234; the velocity V ofthe flow 242 of fluidic medium 180 in the microfluidic channel 122; theviscosity of the first medium 180 and/or the second medium 248, or thelike.

In some embodiments, the dimensions of the microfluidic channel 122 andsequestration pens 224, 226, 228 can be oriented as follows with respectto the vector of the flow 242 of fluidic medium 180 in the microfluidicchannel 122: the microfluidic channel width W_(ch) (or cross-sectionalarea of the microfluidic channel 122) can be substantially perpendicularto the flow 242 of medium 180; the width W_(con) (or cross-sectionalarea) of the connection region 236 at opening 234 can be substantiallyparallel to the flow 242 of medium 180 in the microfluidic channel 122;and/or the length L_(con) of the connection region can be substantiallyperpendicular to the flow 242 of medium 180 in the microfluidic channel122. The foregoing are examples only, and the relative position of themicrofluidic channel 122 and sequestration pens 224, 226, 228 can be inother orientations with respect to each other.

As illustrated in FIG. 2C, the width W_(con) of the connection region236 can be uniform from the proximal opening 234 to the distal opening238. The width W_(con) of the connection region 236 at the distalopening 238 can thus be in any of the ranges identified herein for thewidth W_(con) of the connection region 236 at the proximal opening 234.Alternatively, the width W_(con) of the connection region 236 at thedistal opening 238 can be larger than the width W_(con) of theconnection region 236 at the proximal opening 234.

As illustrated in FIG. 2C, the width of the isolation region 240 at thedistal opening 238 can be substantially the same as the width W_(con) ofthe connection region 236 at the proximal opening 234. The width of theisolation region 240 at the distal opening 238 can thus be in any of theranges identified herein for the width W_(con) of the connection region236 at the proximal opening 234. Alternatively, the width of theisolation region 240 at the distal opening 238 can be larger or smallerthan the width W_(con) of the connection region 236 at the proximalopening 234. Moreover, the distal opening 238 may be smaller than theproximal opening 234 and the width W_(con) of the connection region 236may be narrowed between the proximal opening 234 and distal opening 238.For example, the connection region 236 may be narrowed between theproximal opening and the distal opening, using a variety of differentgeometries (e.g. chamfering the connection region, beveling theconnection region). Further, any part or subpart of the connectionregion 236 may be narrowed (e.g. a portion of the connection regionadjacent to the proximal opening 234).

FIGS. 2D-2F depict another exemplary embodiment of a microfluidic device250 containing a microfluidic circuit 262 and flow channels 264, whichare variations of the respective microfluidic device 100, circuit 132and channel 134 of FIG. 1A. The microfluidic device 250 also has aplurality of sequestration pens 266 that are additional variations ofthe above-described sequestration pens 124, 126, 128, 130, 224, 226 or228. In particular, it should be appreciated that the sequestration pens266 of device 250 shown in FIGS. 2D-2F can replace any of theabove-described sequestration pens 124, 126, 128, 130, 224, 226 or 228in devices 100, 200, 230, 250, 280, 290, 500, 550, 560, 600, 620, 640,670, 700, 720, 720, 750, 760, 780, 808, 810, 812, 900, 1000, 1100, 1200,1300, 1400, 1500. Likewise, the microfluidic device 250 is anothervariant of the microfluidic device 100, and may also have the same or adifferent DEP configuration as the above-described microfluidic device100, 200, 230, 250, 280, 290, 500, 550, 560, 600, 620, 640, 670, 700,720, 720, 750, 760, 780, 808, 810, 812, 900, 1000, 1100, 1200, 1300,1400, 1500 as well as any of the other microfluidic system componentsdescribed herein.

The microfluidic device 250 of FIGS. 2D-2F comprises a support structure(not visible in FIGS. 2D-2F, but can be the same or generally similar tothe support structure 104 of device 100 depicted in FIG. 1A), amicrofluidic circuit structure 256, and a cover (not visible in FIGS.2D-2F, but can be the same or generally similar to the cover 122 ofdevice 100 depicted in FIG. 1A). The microfluidic circuit structure 256includes a frame 252 and microfluidic circuit material 260, which can bethe same as or generally similar to the frame 114 and microfluidiccircuit material 116 of device 100 shown in FIG. 1A. As shown in FIG.2D, the microfluidic circuit 262 defined by the microfluidic circuitmaterial 260 can include multiple channels 264 (two are shown but therecan be more) to which multiple sequestration pens 266 are fluidicallyconnected.

Each sequestration pen 266 can comprise an isolation structure 272, anisolation region 270 within the isolation structure 272, and aconnection region 268. From a proximal opening 274 at the microfluidicchannel 264 to a distal opening 276 at the isolation structure 272, theconnection region 268 fluidically connects the microfluidic channel 264to the isolation region 270. Generally, in accordance with the abovediscussion of FIGS. 2B and 2C, a flow 278 of a first fluidic medium 254in a channel 264 can create secondary flows 282 of the first medium 254from the microfluidic channel 264 into and/or out of the respectiveconnection regions 268 of the sequestration pens 266.

As illustrated in FIG. 2E, the connection region 268 of eachsequestration pen 266 generally includes the area extending between theproximal opening 274 to a channel 264 and the distal opening 276 to anisolation structure 272. The length L_(con) of the connection region 268can be greater than the maximum penetration depth D_(p) of secondaryflow 282, in which case the secondary flow 282 will extend into theconnection region 268 without being redirected toward the isolationregion 270 (as shown in FIG. 2D). Alternatively, at illustrated in FIG.2F, the connection region 268 can have a length L_(con) that is lessthan the maximum penetration depth D_(p), in which case the secondaryflow 282 will extend through the connection region 268 and be redirectedtoward the isolation region 270. In this latter situation, the sum oflengths L_(c1) and L_(c2) of connection region 268 is greater than themaximum penetration depth D_(p), so that secondary flow 282 will notextend into isolation region 270. Whether length L_(con) of connectionregion 268 is greater than the penetration depth D_(p), or the sum oflengths L_(c1) and L_(c2) of connection region 268 is greater than thepenetration depth D_(p), a flow 278 of a first medium 254 in channel 264that does not exceed a maximum velocity V_(max) will produce a secondaryflow having a penetration depth D_(p), and micro-objects (not shown butcan be the same or generally similar to the micro-objects 246 shown inFIG. 2C) in the isolation region 270 of a sequestration pen 266 will notbe drawn out of the isolation region 270 by a flow 278 of first medium254 in channel 264. Nor will the flow 278 in channel 264 drawmiscellaneous materials (not shown) from channel 264 into the isolationregion 270 of a sequestration pen 266. As such, diffusion is the onlymechanism by which components in a first medium 254 in the microfluidicchannel 264 can move from the microfluidic channel 264 into a secondmedium 258 in an isolation region 270 of a sequestration pen 266.Likewise, diffusion is the only mechanism by which components in asecond medium 258 in an isolation region 270 of a sequestration pen 266can move from the isolation region 270 to a first medium 254 in themicrofluidic channel 264. The first medium 254 can be the same medium asthe second medium 258, or the first medium 254 can be a different mediumthan the second medium 258. Alternatively, the first medium 254 and thesecond medium 258 can start out being the same, then become different,e.g., through conditioning of the second medium by one or more cells inthe isolation region 270, or by changing the medium flowing through themicrofluidic channel 264.

As illustrated in FIG. 2E, the width W_(ch) of the microfluidic channels264 (i.e., taken transverse to the direction of a fluid medium flowthrough the microfluidic channel indicated by arrows 278 in FIG. 2D) inthe microfluidic channel 264 can be substantially perpendicular to awidth W_(con1) of the proximal opening 274 and thus substantiallyparallel to a width W_(con2) of the distal opening 276. The widthW_(con1) of the proximal opening 274 and the width W_(con2) of thedistal opening 276, however, need not be substantially perpendicular toeach other. For example, an angle between an axis (not shown) on whichthe width W_(con1) of the proximal opening 274 is oriented and anotheraxis on which the width W_(con2) of the distal opening 276 is orientedcan be other than perpendicular and thus other than 90°. Examples ofalternatively oriented angles include angles in any of the followingranges: from about 30° to about 90°, from about 45° to about 90°, fromabout 60° to about 90°, or the like.

In various embodiments of sequestration pens (e.g. 124, 126, 128, 130,224, 226, 228, or 266), the isolation region (e.g. 240 or 270) isconfigured to contain a plurality of micro-objects. In otherembodiments, the isolation region can be configured to contain only one,two, three, four, five, or a similar relatively small number ofmicro-objects. Accordingly, the volume of an isolation region can be,for example, at least 1×10⁶, 2×10⁶, 4×10⁶, 6×10⁶ cubic microns, or more.

In various embodiments of sequestration pens, the width W_(ch) of themicrofluidic channel (e.g., 122) at a proximal opening (e.g. 234) can bewithin any of the following ranges: about 50-1000 microns, 50-500microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns,50-150 microns, 50-100 microns, 70-500 microns, 70-400 microns, 70-300microns, 70-250 microns, 70-200 microns, 70-150 microns, 90-400 microns,90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns, 100-300microns, 100-250 microns, 100-200 microns, 100-150 microns, and 100-120microns. In some other embodiments, the width W_(ch) of the microfluidicchannel (e.g., 122) at a proximal opening (e.g. 234) can be in a rangeof about 200-800 microns, 200-700 microns, or 200-600 microns. Theforegoing are examples only, and the width W_(ch) of the microfluidicchannel 122 can be in other ranges (e.g., a range defined by any of theendpoints listed above). Moreover, the W_(ch) of the microfluidicchannel 122 can be selected to be in any of these ranges in regions ofthe microfluidic channel other than at a proximal opening of asequestration pen.

In some embodiments, a sequestration pen has a height of about 30 toabout 200 microns, or about 50 to about 150 microns. In someembodiments, the sequestration pen has a cross-sectional area of about1×10⁴-3×10⁶ square microns, 2×10⁴-2×10⁶ square microns, 4×10⁴-1×10⁶square microns, 2×10⁴-5×10⁵ square microns, 2×10⁴-1×10⁵ square micronsor about 2×10⁵-2×10⁶ square microns.

In various embodiments of sequestration pens, the height H_(ch) of themicrofluidic channel (e.g., 122) at a proximal opening (e.g., 234) canbe within any of the following ranges: 20-100 microns, 20-90 microns,20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns,30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70microns, 40-60 microns, or 40-50 microns. The foregoing are examplesonly, and the height H_(ch) of the microfluidic channel (e.g., 122) canbe in other ranges (e.g., a range defined by any of the endpoints listedabove). The height H_(ch) of the microfluidic channel 122 can beselected to be in any of these ranges in regions of the microfluidicchannel other than at a proximal opening of an sequestration pen.

In various embodiments of sequestration pens a cross-sectional area ofthe microfluidic channel (e.g., 122) at a proximal opening (e.g., 234)can be within any of the following ranges: 500-50,000 square microns,500-40,000 square microns, 500-30,000 square microns, 500-25,000 squaremicrons, 500-20,000 square microns, 500-15,000 square microns,500-10,000 square microns, 500-7,500 square microns, 500-5,000 squaremicrons, 1,000-25,000 square microns, 1,000-20,000 square microns,1,000-15,000 square microns, 1,000-10,000 square microns, 1,000-7,500square microns, 1,000-5,000 square microns, 2,000-20,000 square microns,2,000-15,000 square microns, 2,000-10,000 square microns, 2,000-7,500square microns, 2,000-6,000 square microns, 3,000-20,000 square microns,3,000-15,000 square microns, 3,000-10,000 square microns, 3,000-7,500square microns, or 3,000 to 6,000 square microns. The foregoing areexamples only, and the cross-sectional area of the microfluidic channel(e.g., 122) at a proximal opening (e.g., 234) can be in other ranges(e.g., a range defined by any of the endpoints listed above).

In various embodiments of sequestration pens, the length L_(con) of theconnection region (e.g., 236) can be in any of the following ranges:about 1-600 microns, 5-550 microns, 10-500 microns, 15-400 microns,20-300 microns, 20-500 microns, 40-400 microns, 60-300 microns, 80-200microns, or about 100-150 microns. The foregoing are examples only, andlength L_(con) of a connection region (e.g., 236) can be in a differentrange than the foregoing examples (e.g., a range defined by any of theendpoints listed above).

In various embodiments of sequestration pens the width W_(con) of aconnection region (e.g., 236) at a proximal opening (e.g., 234) can bein any of the following ranges: 20-500 microns, 20-400 microns, 20-300microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns,20-60 microns, 30-400 microns, 30-300 microns, 30-200 microns, 30-150microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns,40-200 microns, 40-150 microns, 40-100 microns, 40-80 microns, 40-60microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns,50-80 microns, 60-200 microns, 60-150 microns, 60-100 microns, 60-80microns, 70-150 microns, 70-100 microns, and 80-100 microns. Theforegoing are examples only, and the width W_(con) of a connectionregion (e.g., 236) at a proximal opening (e.g., 234) can be differentthan the foregoing examples (e.g., a range defined by any of theendpoints listed above).

In various embodiments of sequestration pens, the width W_(con) of aconnection region (e.g., 236) at a proximal opening (e.g., 234) can beat least as large as the largest dimension of a micro-object (e.g.,biological cell which may be a T cell, B cell, or an ovum or embryo)that the sequestration pen is intended for. For example, the widthW_(con) of a connection region 236 at a proximal opening 234 of ansequestration pen that an oocyte, ovum, or embryo will be placed intocan be in any of the following ranges: about 100 microns, about 110microns, about 120 microns, about 130 microns, about 140 microns, about150 microns, about 160 microns, about 170 microns, about 180 microns,about 190 microns, about 200 microns, about 225 microns, about 250microns, about 300 microns or about 100-400 microns, about 120-350microns, about 140-300 microns, or about 140-200 microns. The foregoingare examples only, and the width W_(con) of a connection region (e.g.,236) at a proximal opening (e.g., 234) can be different than theforegoing examples (e.g., a range defined by any of the endpoints listedabove).

In various embodiments of sequestration pens, the width W_(pr) of aproximal opening of a connection region may be at least as large as thelargest dimension of a micro-object (e.g., a biological micro-objectsuch as a cell) that the sequestration pen is intended for. For example,the width W_(pr) may be about 50 microns, about 60 microns, about 100microns, about 200 microns, about 300 microns or may be in a range ofabout 50-300 microns, about 50-200 microns, about 50-100 microns, about75-150 microns, about 75-100 microns, or about 200-300 microns

In various embodiments of sequestration pens, a ratio of the lengthL_(con) of a connection region (e.g., 236) to a width Won of theconnection region (e.g., 236) at the proximal opening 234 can be greaterthan or equal to any of the following ratios: 0.5, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or more. Theforegoing are examples only, and the ratio of the length L_(con) of aconnection region 236 to a width W_(con) of the connection region 236 atthe proximal opening 234 can be different than the foregoing examples.

In various embodiments of microfluidic devices 100, 200, 230, 250, 280,290, 500, 550, 560, 600, 620, 640, 670, 700, 720, 720, 750, 760, 780,808, 810, 812, 900, 1000, 1100, 1200, 1300, 1400, 1500, V_(max) can beset around 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, or 1.5 microliters/sec.

In various embodiments of microfluidic devices having sequestrationpens, the volume of an isolation region (e.g., 240) of a sequestrationpen can be, for example, at least 5×10⁵, 8×10⁵, 1×10⁶, 2×10⁶, 4×10⁶,6×10⁶, 8×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, or 8×10⁸ cubic microns, ormore. In various embodiments of microfluidic devices havingsequestration pens, the volume of a sequestration pen may be about5×10⁵, 6×10⁵, 8×10⁵, 1×10⁶, 2×10⁶, 4×10⁶, 8×10⁶, 1×10⁷, 3×10⁷, 5×10⁷, orabout 8×10⁷ cubic microns, or more. In some other embodiments, thevolume of a sequestration pen may be about 1 nanoliter to about 50nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters to about20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about 2nanoliters to about 10 nanoliters.

In various embodiment, the microfluidic device has sequestration pensconfigured as in any of the embodiments discussed herein where themicrofluidic device has about 5 to about 10 sequestration pens, about 10to about 50 sequestration pens, about 100 to about 500 sequestrationpens; about 200 to about 1000 sequestration pens, about 500 to about1500 sequestration pens, about 1000 to about 2000 sequestration pens, orabout 1000 to about 3500 sequestration pens. The sequestration pens neednot all be the same size and may include a variety of configurations(e.g., different widths, different features within the sequestrationpen).

FIG. 2G illustrates a microfluidic device 280 according to oneembodiment. The microfluidic device 280 illustrated in FIG. 2G is astylized diagram of a microfluidic device 100. In practice themicrofluidic device 280 and its constituent circuit elements (e.g.channels 122 and sequestration pens 128) would have the dimensionsdiscussed herein. The microfluidic circuit 120 illustrated in FIG. 2Ghas two ports 107, four distinct channels 122 and four distinct flowpaths 106. The microfluidic device 280 further includes a plurality ofsequestration pens opening off of each channel 122. In the microfluidicdevice illustrated in FIG. 2G, the sequestration pens have a geometrysimilar to the pens illustrated in FIG. 2C and thus, have bothconnection regions and isolation regions. Accordingly, the microfluidiccircuit 120 includes both swept regions (e.g. channels 122 and portionsof the connection regions 236 within the maximum penetration depth D_(p)of the secondary flow 244) and non-swept regions (e.g. isolation regions240 and portions of the connection regions 236 not within the maximumpenetration depth D_(p) of the secondary flow 244).

Coating solutions and coating agents. Without intending to be limited bytheory, maintenance of a biological micro-object (e.g., a biologicalcell) within a microfluidic device (e.g., a DEP-configured and/orEW-configured microfluidic device) may be facilitated (i.e., thebiological micro-object exhibits increased viability, greater expansionand/or greater portability within the microfluidic device) when at leastone or more inner surfaces of the microfluidic device have beenconditioned or coated so as to present a layer of organic and/orhydrophilic molecules that provides the primary interface between themicrofluidic device and biological micro-object(s) maintained therein.In some embodiments, one or more of the inner surfaces of themicrofluidic device (e.g. the inner surface of the electrode activationsubstrate of a DEP-configured microfluidic device, the cover of themicrofluidic device, and/or the surfaces of the circuit material) may betreated with or modified by a coating solution and/or coating agent togenerate the desired layer of organic and/or hydrophilic molecules.

The coating may be applied before or after introduction of biologicalmicro-object(s), or may be introduced concurrently with the biologicalmicro-object(s). In some embodiments, the biological micro-object(s) maybe imported into the microfluidic device in a fluidic medium thatincludes one or more coating agents. In other embodiments, the innersurface(s) of the microfluidic device (e.g., a DEP-configuredmicrofluidic device) are treated or “primed” with a coating solutioncomprising a coating agent prior to introduction of the biologicalmicro-object(s) into the microfluidic device.

In some embodiments, at least one surface of the microfluidic deviceincludes a coating material that provides a layer of organic and/orhydrophilic molecules suitable for maintenance and/or expansion ofbiological micro-object(s) (e.g. provides a conditioned surface asdescribed below). In some embodiments, substantially all the innersurfaces of the microfluidic device include the coating material. Thecoated inner surface(s) may include the surface of a flow region (e.g.,channel), chamber, or sequestration pen, or a combination thereof. Insome embodiments, each of a plurality of sequestration pens has at leastone inner surface coated with coating materials. In other embodiments,each of a plurality of flow regions or channels has at least one innersurface coated with coating materials. In some embodiments, at least oneinner surface of each of a plurality of sequestration pens and each of aplurality of channels is coated with coating materials.

Coating agent/Solution. Any convenient coating agent/coating solutioncan be used, including but not limited to: serum or serum factors,bovine serum albumin (BSA), polymers, detergents, enzymes, and anycombination thereof.

Polymer-based coating materials. The at least one inner surface mayinclude a coating material that comprises a polymer. The polymer may becovalently or non-covalently bound (or may be non-specifically adhered)to the at least one surface. The polymer may have a variety ofstructural motifs, such as found in block polymers (and copolymers),star polymers (star copolymers), and graft or comb polymers (graftcopolymers), all of which may be suitable for the methods disclosedherein.

The polymer may include a polymer including alkylene ether moieties. Awide variety of alkylene ether containing polymers may be suitable foruse in the microfluidic devices described herein. One non-limitingexemplary class of alkylene ether containing polymers are amphiphilicnonionic block copolymers which include blocks of polyethylene oxide(PEO) and polypropylene oxide (PPO) subunits in differing ratios andlocations within the polymer chain. Pluronic® polymers (BASF) are blockcopolymers of this type and are known in the art to be suitable for usewhen in contact with living cells. The polymers may range in averagemolecular mass M_(w) from about 2000 Da to about 20 KDa. In someembodiments, the PEO-PPO block copolymer can have ahydrophilic-lipophilic balance (HLB) greater than about 10 (e.g. 12-18).Specific Pluronic® polymers useful for yielding a coated surface includePluronic® L44, L64, P85, and F127 (including F127NF). Another class ofalkylene ether containing polymers is polyethylene glycol (PEGM_(w)<100,000 Da) or alternatively polyethylene oxide (PEO,M_(w)>100,000). In some embodiments, a PEG may have an M_(w) of about1000 Da, 5000 Da, 10,000 Da or 20,000 Da.

In other embodiments, the coating material may include a polymercontaining carboxylic acid moieties. The carboxylic acid subunit may bean alkyl, alkenyl or aromatic moiety containing subunit. Onenon-limiting example is polylactic acid (PLA). In other embodiments, thecoating material may include a polymer containing phosphate moieties,either at a terminus of the polymer backbone or pendant from thebackbone of the polymer. In yet other embodiments, the coating materialmay include a polymer containing sulfonic acid moieties. The sulfonicacid subunit may be an alkyl, alkenyl or aromatic moiety containingsubunit. One non-limiting example is polystyrene sulfonic acid (PSSA) orpolyanethole sulfonic acid. In further embodiments, the coating materialmay include a polymer including amine moieties. The polyamino polymermay include a natural polyamine polymer or a synthetic polyaminepolymer. Examples of natural polyamines include spermine, spermidine,and putrescine.

In other embodiments, the coating material may include a polymercontaining saccharide moieties. In a non-limiting example,polysaccharides such as xanthan gum or dextran may be suitable to form amaterial which may reduce or prevent cell sticking in the microfluidicdevice. For example, a dextran polymer having a size about 3 kDa may beused to provide a coating material for a surface within a microfluidicdevice.

In other embodiments, the coating material may include a polymercontaining nucleotide moieties, i.e. a nucleic acid, which may haveribonucleotide moieties or deoxyribonucleotide moieties, providing apolyelectrolyte surface. The nucleic acid may contain only naturalnucleotide moieties or may contain unnatural nucleotide moieties whichcomprise nucleobase, ribose or phosphate moiety analogs such as7-deazaadenine, pentose, methyl phosphonate or phosphorothioate moietieswithout limitation.

In yet other embodiments, the coating material may include a polymercontaining amino acid moieties. The polymer containing amino acidmoieties may include a natural amino acid containing polymer or anunnatural amino acid containing polymer, either of which may include apeptide, a polypeptide or a protein. In one non-limiting example, theprotein may be bovine serum albumin (BSA) and/or serum (or a combinationof multiple different sera) comprising albumin and/or one or more othersimilar proteins as coating agents. The serum can be from any convenientsource, including but not limited to fetal calf serum, sheep serum, goatserum, horse serum, and the like. In certain embodiments, BSA in acoating solution is present in a range of form about 1 mg/mL to about100 mg/mL, including 5 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, or more or anywhere inbetween. In certain embodiments, serum in a coating solution may bepresent in a range of from about 20% (v/v) to about 50% v/v, including25%, 30%, 35%, 40%, 45%, or more or anywhere in between. In someembodiments, BSA may be present as a coating agent in a coating solutionat 5 mg/mL, whereas in other embodiments, BSA may be present as acoating agent in a coating solution at 70 mg/mL. In certain embodiments,serum is present as a coating agent in a coating solution at 30%. Insome embodiments, an extracellular matrix (ECM) protein may be providedwithin the coating material for optimized cell adhesion to foster cellgrowth. A cell matrix protein, which may be included in a coatingmaterial, can include, but is not limited to, a collagen, an elastin, anRGD-containing peptide (e.g. a fibronectin), or a laminin. In yet otherembodiments, growth factors, cytokines, hormones or other cell signalingspecies may be provided within the coating material of the microfluidicdevice.

In some embodiments, the coating material may include a polymercontaining more than one of alkylene oxide moieties, carboxylic acidmoieties, sulfonic acid moieties, phosphate moieties, saccharidemoieties, nucleotide moieties, or amino acid moieties. In otherembodiments, the polymer conditioned surface may include a mixture ofmore than one polymer each having alkylene oxide moieties, carboxylicacid moieties, sulfonic acid moieties, phosphate moieties, saccharidemoieties, nucleotide moieties, and/or amino acid moieties, which may beindependently or simultaneously incorporated into the coating material.

Covalently linked coating materials. In some embodiments, the at leastone inner surface includes covalently linked molecules that provide alayer of organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) within themicrofluidic device, providing a conditioned surface for such cells.

The covalently linked molecules include a linking group, wherein thelinking group is covalently linked to one or more surfaces of themicrofluidic device, as described below. The linking group is alsocovalently linked to a moiety configured to provide a layer of organicand/or hydrophilic molecules suitable for maintenance/expansion ofbiological micro-object(s).

In some embodiments, the covalently linked moiety configured to providea layer of organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) may include alkyl orfluoroalkyl (which includes perfluoroalkyl) moieties; mono- orpolysaccharides (which may include but is not limited to dextran);alcohols (including but not limited to propargyl alcohol); polyalcohols,including but not limited to polyvinyl alcohol; alkylene ethers,including but not limited to polyethylene glycol; polyelectrolytes(including but not limited to polyacrylic acid or polyvinyl phosphonicacid); amino groups (including derivatives thereof, such as, but notlimited to alkylated amines, hydroxyalkylated amino group, guanidinium,and heterocylic groups containing an unaromatized nitrogen ring atom,such as, but not limited to morpholinyl or piperazinyl); carboxylicacids including but not limited to propiolic acid (which may provide acarboxylate anionic surface); phosphonic acids, including but notlimited to ethynyl phosphonic acid (which may provide a phosphonateanionic surface); sulfonate anions; carboxybetaines; sulfobetaines;sulfamic acids; or amino acids.

In various embodiments, the covalently linked moiety configured toprovide a layer of organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) in the microfluidicdevice may include non-polymeric moieties such as an alkyl moiety, asubstituted alkyl moiety, such as a fluoroalkyl moiety (including butnot limited to a perfluoroalkyl moiety), amino acid moiety, alcoholmoiety, amino moiety, carboxylic acid moiety, phosphonic acid moiety,sulfonic acid moiety, sulfamic acid moiety, or saccharide moiety.Alternatively, the covalently linked moiety may include polymericmoieties, which may be any of the moieties described above.

In some embodiments, the covalently linked alkyl moiety may comprisecarbon atoms forming a linear chain (e.g., a linear chain of at least 10carbons, or at least 14, 16, 18, 20, 22, or more carbons) and may be anunbranched alkyl moiety. In some embodiments, the alkyl group mayinclude a substituted alkyl group (e.g., some of the carbons in thealkyl group can be fluorinated or perfluorinated). In some embodiments,the alkyl group may include a first segment, which may include aperfluoroalkyl group, joined to a second segment, which may include anon-substituted alkyl group, where the first and second segments may bejoined directly or indirectly (e.g., by means of an ether linkage). Thefirst segment of the alkyl group may be located distal to the linkinggroup, and the second segment of the alkyl group may be located proximalto the linking group.

In other embodiments, the covalently linked moiety may include at leastone amino acid, which may include more than one type of amino acid.Thus, the covalently linked moiety may include a peptide or a protein.In some embodiments, the covalently linked moiety may include an aminoacid which may provide a zwitterionic surface to support cell growth,viability, portability, or any combination thereof.

In other embodiments, the covalently linked moiety may include at leastone alkylene oxide moiety, and may include any alkylene oxide polymer asdescribed above. One useful class of alkylene ether containing polymersis polyethylene glycol (PEG M_(w)<100,000 Da) or alternativelypolyethylene oxide (PEO, M_(w)>100,000). In some embodiments, a PEG mayhave an M_(w) of about 1000 Da, 5000 Da, 10,000 Da or 20,000 Da.

The covalently linked moiety may include one or more saccharides. Thecovalently linked saccharides may be mono-, di-, or polysaccharides. Thecovalently linked saccharides may be modified to introduce a reactivepairing moiety which permits coupling or elaboration for attachment tothe surface. Exemplary reactive pairing moieties may include aldehyde,alkyne or halo moieties. A polysaccharide may be modified in a randomfashion, wherein each of the saccharide monomers may be modified or onlya portion of the saccharide monomers within the polysaccharide aremodified to provide a reactive pairing moiety that may be coupleddirectly or indirectly to a surface. One exemplar may include a dextranpolysaccharide, which may be coupled indirectly to a surface via anunbranched linker.

The covalently linked moiety may include one or more amino groups. Theamino group may be a substituted amine moiety, guanidine moiety,nitrogen-containing heterocyclic moiety or heteroaryl moiety. The aminocontaining moieties may have structures permitting pH modification ofthe environment within the microfluidic device, and optionally, withinthe sequestration pens and/or flow regions (e.g., channels).

The coating material providing a conditioned surface may comprise onlyone kind of covalently linked moiety or may include more than onedifferent kind of covalently linked moiety. For example, the fluoroalkylconditioned surfaces (including perfluoroalkyl) may have a plurality ofcovalently linked moieties which are all the same, e.g., having the samelinking group and covalent attachment to the surface, the same overalllength, and the same number of fluoromethylene units comprising thefluoroalkyl moiety. Alternatively, the coating material may have morethan one kind of covalently linked moiety attached to the surface. Forexample, the coating material may include molecules having covalentlylinked alkyl or fluoroalkyl moieties having a specified number ofmethylene or fluoromethylene units and may further include a further setof molecules having charged moieties covalently attached to an alkyl orfluoroalkyl chain having a greater number of methylene orfluoromethylene units, which may provide capacity to present bulkiermoieties at the coated surface. In this instance, the first set ofmolecules having different, less sterically demanding termini and fewerbackbone atoms can help to functionalize the entire substrate surfaceand thereby prevent undesired adhesion or contact with thesilicon/silicon oxide, hafnium oxide or alumina making up the substrateitself. In another example, the covalently linked moieties may provide azwitterionic surface presenting alternating charges in a random fashionon the surface.

Conditioned surface properties. Aside from the composition of theconditioned surface, other factors such as physical thickness of thehydrophobic material can impact DEP force. Various factors can alter thephysical thickness of the conditioned surface, such as the manner inwhich the conditioned surface is formed on the substrate (e.g. vapordeposition, liquid phase deposition, spin coating, flooding, andelectrostatic coating). In some embodiments, the conditioned surface hasa thickness in the range of about 1 nm to about 10 nm; about 1 nm toabout 7 nm; about 1 nm to about 5 nm; or any individual valuetherebetween. In other embodiments, the conditioned surface formed bythe covalently linked moieties may have a thickness of about 10 nm toabout 50 nm. In various embodiments, the conditioned surface prepared asdescribed herein has a thickness of less than 10 nm. In someembodiments, the covalently linked moieties of the conditioned surfacemay form a monolayer when covalently linked to the surface of themicrofluidic device (e.g., a DEP configured substrate surface) and mayhave a thickness of less than 10 nm (e.g., less than 5 nm, or about 1.5to 3.0 nm). These values are in contrast to that of a CYTOP® (AsahiGlass Co., Ltd. JP) fluoropolymer spin coating, which has a thickness inthe range of about 30 nm. In some embodiments, the conditioned surfacedoes not require a perfectly formed monolayer to be suitably functionalfor operation within a DEP-configured microfluidic device.

In various embodiments, the coating material providing a conditionedsurface of the microfluidic device may provide desirable electricalproperties. Without intending to be limited by theory, one factor thatimpacts robustness of a surface coated with a particular coatingmaterial is intrinsic charge trapping. Different coating materials maytrap electrons, which can lead to breakdown of the coating material.Defects in the coating material may increase charge trapping and lead tofurther breakdown of the coating material. Similarly, different coatingmaterials have different dielectric strengths (i.e. the minimum appliedelectric field that results in dielectric breakdown), which may impactcharge trapping. In certain embodiments, the coating material can havean overall structure (e.g., a densely-packed monolayer structure) thatreduces or limits that amount of charge trapping.

In addition to its electrical properties, the conditioned surface mayalso have properties that are beneficial in use with biologicalmolecules. For example, a conditioned surface that contains fluorinated(or perfluorinated) carbon chains may provide a benefit relative toalkyl-terminated chains in reducing the amount of surface fouling.Surface fouling, as used herein, refers to the amount of indiscriminatematerial deposition on the surface of the microfluidic device, which mayinclude permanent or semi-permanent deposition of biomaterials such asprotein and its degradation products, nucleic acids and respectivedegradation products and the like.

Unitary or Multi-part conditioned surface. The covalently linked coatingmaterial may be formed by reaction of a molecule which already containsthe moiety configured to provide a layer of organic and/or hydrophilicmolecules suitable for maintenance/expansion of biologicalmicro-object(s) in the microfluidic device, as is described below.Alternatively, the covalently linked coating material may be formed in atwo-part sequence by coupling the moiety configured to provide a layerof organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) to a surfacemodifying ligand that itself has been covalently linked to the surface.

Methods of preparing a covalently linked coating material. In someembodiments, a coating material that is covalently linked to the surfaceof a microfluidic device (e.g., including at least one surface of thesequestration pens and/or flow regions) has a structure of Formula 1 orFormula 2. When the coating material is introduced to the surface in onestep, it has a structure of Formula 1, while when the coating materialis introduced in a multiple step process, it has a structure of Formula2.

The coating material may be linked covalently to oxides of the surfaceof a DEP-configured or EW-configured substrate. The DEP- orEW-configured substrate may comprise silicon, silicon oxide, alumina, orhafnium oxide. Oxides may be present as part of the native chemicalstructure of the substrate or may be introduced as discussed below.

The coating material may be attached to the oxides via a linking group(“LG”), which may be a siloxy or phosphonate ester group formed from thereaction of a siloxane or phosphonic acid group with the oxides. Themoiety configured to provide a layer of organic and/or hydrophilicmolecules suitable for maintenance/expansion of biologicalmicro-object(s) in the microfluidic device can be any of the moietiesdescribed herein. The linking group LG may be directly or indirectlyconnected to the moiety configured to provide a layer of organic and/orhydrophilic molecules suitable for maintenance/expansion of biologicalmicro-object(s) in the microfluidic device. When the linking group LG isdirectly connected to the moiety, optional linker (“L”) is not presentand n is 0. When the linking group LG is indirectly connected to themoiety, linker L is present and n is 1. The linker L may have a linearportion where a backbone of the linear portion may include 1 to 200non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms, subject to chemicalbonding limitations as is known in the art. It may be interrupted withany combination of one or more moieties selected from the groupconsisting of ether, amino, carbonyl, amido, or phosphonate groups,arylene, heteroarylene, or heterocyclic groups. In some embodiments, thebackbone of the linker L may include 10 to 20 atoms. In otherembodiments, the backbone of the linker L may include about 5 atoms toabout 200 atoms; about 10 atoms to about 80 atoms; about 10 atoms toabout 50 atoms; or about 10 atoms to about 40 atoms. In someembodiments, the backbone atoms are all carbon atoms.

In some embodiments, the moiety configured to provide a layer of organicand/or hydrophilic molecules suitable for maintenance/expansion ofbiological micro-object(s) may be added to the surface of the substratein a multi-step process, and has a structure of Formula 2, as shownabove. The moiety may be any of the moieties described above.

In some embodiments, the coupling group CG represents the resultantgroup from reaction of a reactive moiety R_(x) and a reactive pairingmoiety R_(px) (i.e., a moiety configured to react with the reactivemoiety R_(x)). For example, one typical coupling group CG may include acarboxamidyl group, which is the result of the reaction of an aminogroup with a derivative of a carboxylic acid, such as an activatedester, an acid chloride or the like. Other CG may include a triazolylenegroup, a carboxamidyl, thioamidyl, an oxime, a mercaptyl, a disulfide,an ether, or alkenyl group, or any other suitable group that may beformed upon reaction of a reactive moiety with its respective reactivepairing moiety. The coupling group CG may be located at the second end(i.e., the end proximal to the moiety configured to provide a layer oforganic and/or hydrophilic molecules suitable for maintenance/expansionof biological micro-object(s) in the microfluidic device) of linker L,which may include any combination of elements as described above. Insome other embodiments, the coupling group CG may interrupt the backboneof the linker L. When the coupling group CG is triazolylene, it may bethe product resulting from a Click coupling reaction and may be furthersubstituted (e.g., a dibenzocylcooctenyl fused triazolylene group).

In some embodiments, the coating material (or surface modifying ligand)is deposited on the inner surfaces of the microfluidic device usingchemical vapor deposition. The vapor deposition process can beoptionally improved, for example, by pre-cleaning the cover 110, themicrofluidic circuit material 116, and/or the substrate (e.g., the innersurface 208 of the electrode activation substrate 206 of aDEP-configured substrate, or a dielectric layer of the support structure104 of an EW-configured substrate), by exposure to a solvent bath,sonication or a combination thereof. Alternatively, or in addition, suchpre-cleaning can include treating the cover 110, the microfluidiccircuit material 116, and/or the substrate in an oxygen plasma cleaner,which can remove various impurities, while at the same time introducingan oxidized surface (e.g. oxides at the surface, which may be covalentlymodified as described herein). Alternatively, liquid-phase treatments,such as a mixture of hydrochloric acid and hydrogen peroxide or amixture of sulfuric acid and hydrogen peroxide (e.g., piranha solution,which may have a ratio of sulfuric acid to hydrogen peroxide in a rangefrom about 3:1 to about 7:1) may be used in place of an oxygen plasmacleaner.

In some embodiments, vapor deposition is used to coat the inner surfacesof the microfluidic device 200 after the microfluidic device 200 hasbeen assembled to form an enclosure 102 defining a microfluidic circuit120. Without intending to be limited by theory, depositing such acoating material on a fully-assembled microfluidic circuit 120 may bebeneficial in preventing delamination caused by a weakened bond betweenthe microfluidic circuit material 116 and the electrode activationsubstrate 206 dielectric layer and/or the cover 110. In embodimentswhere a two-step process is employed the surface modifying ligand may beintroduced via vapor deposition as described above, with subsequentintroduction of the moiety configured provide a layer of organic and/orhydrophilic molecules suitable for maintenance/expansion of biologicalmicro-object(s). The subsequent reaction may be performed by exposingthe surface modified microfluidic device to a suitable coupling reagentin solution.

FIG. 2H depicts a cross-sectional view of a microfluidic device 290having an exemplary covalently linked coating material providing aconditioned surface. As illustrated, the coating materials 298 (shownschematically) can comprise a monolayer of densely-packed moleculescovalently bound to both the inner surface 294 of a base 286, which maybe a DEP substrate, and the inner surface 292 of a cover 288 of themicrofluidic device 290. The coating material 298 can be disposed onsubstantially all inner surfaces 294, 292 proximal to, and facinginwards towards, the enclosure 284 of the microfluidic device 290,including, in some embodiments and as discussed above, the surfaces ofmicrofluidic circuit material (not shown) used to define circuitelements and/or structures within the microfluidic device 290. Inalternate embodiments, the coating material 298 can be disposed on onlyone or some of the inner surfaces of the microfluidic device 290.

In the embodiment shown in FIG. 2H, the coating material 298 can includea monolayer of organosiloxane molecules, each molecule covalently bondedto the inner surfaces 292, 294 of the microfluidic device 290 via asiloxy linker 296. Any of the above-discussed coating materials 298 canbe used (e.g. an alkyl-terminated, a fluoroalkyl terminated moiety, aPEG-terminated moiety, a dextran terminated moiety, or a terminal moietycontaining positive or negative charges for the organosiloxy moieties),where the terminal moiety is disposed at its enclosure-facing terminus(i.e. the portion of the monolayer of the coating material 298 that isnot bound to the inner surfaces 292, 294 and is proximal to theenclosure 284).

In other embodiments, the coating material 298 used to coat the innersurface(s) 292, 294 of the microfluidic device 290 can include anionic,cationic, or zwitterionic moieties, or any combination thereof. Withoutintending to be limited by theory, by presenting cationic moieties,anionic moieties, and/or zwitterionic moieties at the inner surfaces ofthe enclosure 284 of the microfluidic circuit 120, the coating material298 can form strong hydrogen bonds with water molecules such that theresulting water of hydration acts as a layer (or “shield”) thatseparates the biological micro-objects from interactions withnon-biological molecules (e.g., the silicon and/or silicon oxide of thesubstrate). In addition, in embodiments in which the coating material298 is used in conjunction with coating agents, the anions, cations,and/or zwitterions of the coating material 298 can form ionic bonds withthe charged portions of non-covalent coating agents (e.g. proteins insolution) that are present in a medium 180 (e.g. a coating solution) inthe enclosure 284.

In still other embodiments, the coating material may comprise or bechemically modified to present a hydrophilic coating agent at itsenclosure-facing terminus. In some embodiments, the coating material mayinclude an alkylene ether containing polymer, such as PEG. In someembodiments, the coating material may include a polysaccharide, such asdextran. Like the charged moieties discussed above (e.g., anionic,cationic, and zwitterionic moieties), the hydrophilic coating agent canform strong hydrogen bonds with water molecules such that the resultingwater of hydration acts as a layer (or “shield”) that separates thebiological micro-objects from interactions with non-biological molecules(e.g., the silicon and/or silicon oxide of the substrate). Furtherdetails of appropriate coating treatments and modifications may be foundat U.S. application Ser. No. 15/135,707, filed on Apr. 22, 2016, and isincorporated by reference in its entirety.

Additional system components for maintenance of viability of cellswithin the sequestration pens of the microfluidic device. To promotegrowth and/or expansion of cell populations, environmental conditionsconducive to maintaining functional cells may be provided by additionalcomponents of the system. For example, such additional components canprovide nutrients, cell growth signaling species, pH modulation, gasexchange, temperature control, and removal of waste products from cells.

System Operation and Optical Control. FIGS. 3A through 3B shows variousembodiments of system 150 which can be used to operate and observemicrofluidic devices (e.g. 100, 200, 230, 250, 280, 290, 500, 550, 560,600, 620, 640, 670, 700, 720, 720, 750, 760, 780, 808, 810, 812, 900,1000, 1100, 1200, 1300, 1400, 1500) according to the present disclosure.As illustrated in FIG. 3A, the system 150 can include a structure(“nest”) 300 configured to hold a microfluidic device 100 (not shown),or any other microfluidic device described herein. The nest 300 caninclude a socket 302 capable of interfacing with the microfluidic device320 (e.g., an optically-actuated electrokinetic device 100) andproviding electrical connections from power source 192 to microfluidicdevice 320. The nest 300 can further include an integrated electricalsignal generation subsystem 304. The electrical signal generationsubsystem 304 can be configured to supply a biasing voltage to socket302 such that the biasing voltage is applied across a pair of electrodesin the microfluidic device 320 when it is being held by socket 302.Thus, the electrical signal generation subsystem 304 can be part ofpower source 192. The ability to apply a biasing voltage to microfluidicdevice 320 does not mean that a biasing voltage will be applied at alltimes when the microfluidic device 320 is held by the socket 302.Rather, in most cases, the biasing voltage will be appliedintermittently, e.g., only as needed to facilitate the generation ofelectrokinetic forces, such as dielectrophoresis or electro-wetting, inthe microfluidic device 320.

As illustrated in FIG. 3A, the nest 300 can include a printed circuitboard assembly (PCBA) 322. The electrical signal generation subsystem304 can be mounted on and electrically integrated into the PCBA 322. Theexemplary support includes socket 302 mounted on PCBA 322, as well.

Typically, the electrical signal generation subsystem 304 will include awaveform generator (not shown). The electrical signal generationsubsystem 304 can further include an oscilloscope (not shown) and/or awaveform amplification circuit (not shown) configured to amplify awaveform received from the waveform generator. The oscilloscope, ifpresent, can be configured to measure the waveform supplied to themicrofluidic device 320 held by the socket 302. In certain embodiments,the oscilloscope measures the waveform at a location proximal to themicrofluidic device 320 (and distal to the waveform generator), thusensuring greater accuracy in measuring the waveform actually applied tothe device. Data obtained from the oscilloscope measurement can be, forexample, provided as feedback to the waveform generator, and thewaveform generator can be configured to adjust its output based on suchfeedback. An example of a suitable combined waveform generator andoscilloscope is the Red Pitaya™.

In certain embodiments, the nest 300 further includes a controller 308,such as a microprocessor used to sense and/or control the electricalsignal generation subsystem 304. Examples of suitable microprocessorsinclude the Arduino™ microprocessors, such as the Arduino Nano™. Thecontroller 308 may be used to perform functions and analysis or maycommunicate with an external master controller 154 (shown in FIG. 1A) toperform functions and analysis. In the embodiment illustrated in FIG. 3Athe controller 308 communicates with a master controller 154 through aninterface 310 (e.g., a plug or connector).

In some embodiments, the nest 300 can include an electrical signalgeneration subsystem 304 comprising a Red Pitaya™ waveformgenerator/oscilloscope unit (“Red Pitaya unit”) and a waveformamplification circuit that amplifies the waveform generated by the RedPitaya unit and passes the amplified voltage to the microfluidic device100. In some embodiments, the Red Pitaya unit is configured to measurethe amplified voltage at the microfluidic device 320 and then adjust itsown output voltage as needed such that the measured voltage at themicrofluidic device 320 is the desired value. In some embodiments, thewaveform amplification circuit can have a +6.5V to −6.5V power supplygenerated by a pair of DC-DC converters mounted on the PCBA 322,resulting in a signal of up to 13 Vpp at the microfluidic device 100.

As illustrated in FIG. 3A, the support structure 300 (e.g., nest) canfurther include a thermal control subsystem 306. The thermal controlsubsystem 306 can be configured to regulate the temperature ofmicrofluidic device 320 held by the support structure 300. For example,the thermal control subsystem 306 can include a Peltier thermoelectricdevice (not shown) and a cooling unit (not shown). The Peltierthermoelectric device can have a first surface configured to interfacewith at least one surface of the microfluidic device 320. The coolingunit can be, for example, a cooling block (not shown), such as aliquid-cooled aluminum block. A second surface of the Peltierthermoelectric device (e.g., a surface opposite the first surface) canbe configured to interface with a surface of such a cooling block. Thecooling block can be connected to a fluidic path 314 configured tocirculate cooled fluid through the cooling block. In the embodimentillustrated in FIG. 3A, the support structure 300 includes an inlet 316and an outlet 318 to receive cooled fluid from an external reservoir(not shown), introduce the cooled fluid into the fluidic path 314 andthrough the cooling block, and then return the cooled fluid to theexternal reservoir. In some embodiments, the Peltier thermoelectricdevice, the cooling unit, and/or the fluidic path 314 can be mounted ona casing 312 of the support structure 300. In some embodiments, thethermal control subsystem 306 is configured to regulate the temperatureof the Peltier thermoelectric device so as to achieve a targettemperature for the microfluidic device 320. Temperature regulation ofthe Peltier thermoelectric device can be achieved, for example, by athermoelectric power supply, such as a Pololu™ thermoelectric powersupply (Pololu Robotics and Electronics Corp.). The thermal controlsubsystem 306 can include a feedback circuit, such as a temperaturevalue provided by an analog circuit. Alternatively, the feedback circuitcan be provided by a digital circuit.

In some embodiments, the nest 300 can include a thermal controlsubsystem 306 with a feedback circuit that is an analog voltage dividercircuit (not shown) which includes a resistor (e.g., with resistance 1kOhm+/−0.1%, temperature coefficient +/−0.02 ppm/C0) and a NTCthermistor (e.g., with nominal resistance 1 kOhm+/−0.01%). In someinstances, the thermal control subsystem 306 measures the voltage fromthe feedback circuit and then uses the calculated temperature value asinput to an on-board PID control loop algorithm. Output from the PIDcontrol loop algorithm can drive, for example, both a directional and apulse-width-modulated signal pin on a Pololu™ motor drive (not shown) toactuate the thermoelectric power supply, thereby controlling the Peltierthermoelectric device.

The nest 300 can include a serial port 324 which allows themicroprocessor of the controller 308 to communicate with an externalmaster controller 154 via the interface 310. In addition, themicroprocessor of the controller 308 can communicate (e.g., via a Plinktool (not shown)) with the electrical signal generation subsystem 304and thermal control subsystem 306. Thus, via the combination of thecontroller 308, the interface 310, and the serial port 324, theelectrical signal generation subsystem 304 and the thermal controlsubsystem 306 can communicate with the external master controller 154.In this manner, the master controller 154 can, among other things,assist the electrical signal generation subsystem 304 by performingscaling calculations for output voltage adjustments. A Graphical UserInterface (GUI) (not shown) provided via a display device 170 coupled tothe external master controller 154, can be configured to plottemperature and waveform data obtained from the thermal controlsubsystem 306 and the electrical signal generation subsystem 304,respectively. Alternatively, or in addition, the GUI can allow forupdates to the controller 308, the thermal control subsystem 306, andthe electrical signal generation subsystem 304.

As discussed above, system 150 can include an imaging device 194. Insome embodiments, the imaging device 194 includes a light modulatingsubsystem 330 (See FIG. 3B). The light modulating subsystem 330 caninclude a digital mirror device (DMD) or a microshutter array system(MSA), either of which can be configured to receive light from a lightsource 332 and transmits a subset of the received light into an opticaltrain of microscope 350. Alternatively, the light modulating subsystem330 can include a device that produces its own light (and thus dispenseswith the need for a light source 332), such as an organic light emittingdiode display (OLED), a liquid crystal on silicon (LCOS) device, aferroelectric liquid crystal on silicon device (FLCOS), or atransmissive liquid crystal display (LCD). The light modulatingsubsystem 330 can be, for example, a projector. Thus, the lightmodulating subsystem 330 can be capable of emitting both structured andunstructured light. One example of a suitable light modulating subsystem330 is the Mosaic™ system from Andor Technologies™. In certainembodiments, imaging module 164 and/or motive module 162 of system 150can control the light modulating subsystem 330.

In certain embodiments, the imaging device 194 further includes amicroscope 350. In such embodiments, the nest 300 and light modulatingsubsystem 330 can be individually configured to be mounted on themicroscope 350. The microscope 350 can be, for example, a standardresearch-grade light microscope or fluorescence microscope. Thus, thenest 300 can be configured to be mounted on the stage 344 of themicroscope 350 and/or the light modulating subsystem 330 can beconfigured to mount on a port of microscope 350. In other embodiments,the nest 300 and the light modulating subsystem 330 described herein canbe integral components of microscope 350.

In certain embodiments, the microscope 350 can further include one ormore detectors 348. In some embodiments, the detector 348 is controlledby the imaging module 164. The detector 348 can include an eye piece, acharge-coupled device (CCD), a camera (e.g., a digital camera), or anycombination thereof. If at least two detectors 348 are present, onedetector can be, for example, a fast-frame-rate camera while the otherdetector can be a high sensitivity camera. Furthermore, the microscope350 can include an optical train configured to receive reflected and/oremitted light from the microfluidic device 320 and focus at least aportion of the reflected and/or emitted light on the one or moredetectors 348. The optical train of the microscope can also includedifferent tube lenses (not shown) for the different detectors, such thatthe final magnification on each detector can be different.

In certain embodiments, imaging device 194 is configured to use at leasttwo light sources. For example, a first light source 332 can be used toproduce structured light (e.g., via the light modulating subsystem 330)and a second light source 334 can be used to provide unstructured light.The first light source 332 can produce structured light foroptically-actuated electrokinesis and/or fluorescent excitation, and thesecond light source 334 can be used to provide bright fieldillumination. In these embodiments, the motive module 164 can be used tocontrol the first light source 332 and the imaging module 164 can beused to control the second light source 334. The optical train of themicroscope 350 can be configured to (1) receive structured light fromthe light modulating subsystem 330 and focus the structured light on atleast a first region in a microfluidic device, such as anoptically-actuated electrokinetic device, when the device is being heldby the nest 300, and (2) receive reflected and/or emitted light from themicrofluidic device and focus at least a portion of such reflectedand/or emitted light onto detector 348. The optical train can be furtherconfigured to receive unstructured light from a second light source andfocus the unstructured light on at least a second region of themicrofluidic device, when the device is held by the nest 300. In certainembodiments, the first and second regions of the microfluidic device canbe overlapping regions. For example, the first region can be a subset ofthe second region. In other embodiments, the second light source 334 mayadditionally or alternatively include a laser, which may have anysuitable wavelength of light. The representation of the optical systemshown in FIG. 3B is a schematic representation only, and the opticalsystem may include additional filters, notch filters, lenses and thelike. When the second light source 334 includes one or more lightsource(s) for brightfield and/or fluorescent excitation, as well aslaser illumination the physical arrangement of the light source(s) mayvary from that shown in FIG. 3B, and the laser illumination may beintroduced at any suitable physical location within the optical system.The schematic locations of light source 432 and light source 402/lightmodulating subsystem 404 may be interchanged as well.

In FIG. 3B, the first light source 332 is shown supplying light to alight modulating subsystem 330, which provides structured light to theoptical train of the microscope 350 of system 355 (not shown). Thesecond light source 334 is shown providing unstructured light to theoptical train via a beam splitter 336. Structured light from the lightmodulating subsystem 330 and unstructured light from the second lightsource 334 travel from the beam splitter 336 through the optical traintogether to reach a second beam splitter (or dichroic filter 338,depending on the light provided by the light modulating subsystem 330),where the light gets reflected down through the objective 336 to thesample plane 342. Reflected and/or emitted light from the sample plane342 then travels back up through the objective 340, through the beamsplitter and/or dichroic filter 338, and to a dichroic filter 346. Onlya fraction of the light reaching dichroic filter 346 passes through andreaches the detector 348.

In some embodiments, the second light source 334 emits blue light. Withan appropriate dichroic filter 346, blue light reflected from the sampleplane 342 is able to pass through dichroic filter 346 and reach thedetector 348. In contrast, structured light coming from the lightmodulating subsystem 330 gets reflected from the sample plane 342, butdoes not pass through the dichroic filter 346. In this example, thedichroic filter 346 is filtering out visible light having a wavelengthlonger than 495 nm. Such filtering out of the light from the lightmodulating subsystem 330 would only be complete (as shown) if the lightemitted from the light modulating subsystem did not include anywavelengths shorter than 495 nm. In practice, if the light coming fromthe light modulating subsystem 330 includes wavelengths shorter than 495nm (e.g., blue wavelengths), then some of the light from the lightmodulating subsystem would pass through filter 346 to reach the detector348. In such an embodiment, the filter 346 acts to change the balancebetween the amount of light that reaches the detector 348 from the firstlight source 332 and the second light source 334. This can be beneficialif the first light source 332 is significantly stronger than the secondlight source 334. In other embodiments, the second light source 334 canemit red light, and the dichroic filter 346 can filter out visible lightother than red light (e.g., visible light having a wavelength shorterthan 650 nm).

Devices and Method for dislodging one or more micro-objects usingoptically driven convection and micro-object displacement. Micro-objectssuch as biological cells or embryos, may be moved in their localenvironment, such as within a microfluidic device by a number of forces,including, but not limited to gravity, fluidic flow induced by amechanical pump, electrowetting and/or dielectrophoresis (DEP). In orderto more effectively move micro-objects from one location (e.g. aspecific location where the micro-objects may have been cultured withina microfluidic device) to another location (e.g. another area of thesame microfluidic device or a separate device such as a multiwellplate), varying force vectors may be applied to achieve celltranslocation. While dielectrophoresis (DEP), fluid displacement, andthe like may be sufficient to move cells in the desired manner, forcesapplied at different scale (e.g., a more powerful force or a morelocalized force), in different ways (convective forces, shear flowforces, impacting forces such as cavitation or contact with a meniscusof a bubble, or any combination thereof) and/or on different timescales(e.g., from milliseconds to minutes in duration) may also be employed toassist in moving cells from a present location and/or to a selectedlocation. In one non-limiting example, application of forces other thanDEP may be useful to move biological cells have been cultured within amicrofluidic device for a period of time. The cells may have attached toa surface of the microfluidic device such that DEP forces or gravity maynot be sufficient to move the cells from an attached position.Therefore, forces having other characteristics may be useful indislodging one or more biological cell for which DEP forces are notsufficient or where gravity or mechanically pumped fluidic flow cannotselectively and/or sufficiently dislodge a selected cell.

It has been surprisingly found that optical illumination of discreteselected regions on or within a microfluidic device can heat a portionof a fluidic medium within the microfluidic circuit of the microfluidicdevice to provide a variety of displacement forces differing in scale,physical type and/or timescale which are capable of displacingmicro-objects (including but not limited to biological cells) and/ormixing fluidic media (which may contain micro-objects includingbiological cells) within the microfluidic device, while still providingat least a portion of the micro-objects so displaced are still viable.The generation of such displacing forces may be applied more than onceat the same discrete selected region or adjacent thereto, such thatrepeated force can be applied to dislodge cells and/or mix media (whichmay include micro-objects), while being sufficiently non-destructivetowards micro-objects. Translocating cells from one area, which in someembodiments may be a chamber, sequestration pen, or other microfluidiccircuit element of a microfluidic device, to another area and/orlocation within the microfluidic device, or alternatively, to anotherdevice outside of the microfluidic device (e.g. a multi-well plate) maybe accomplished by applying a pulse of optical illumination to selecteddiscrete regions within the microfluidic device. The pulse of opticalillumination may be applied on, or within proximity to, the cells ofinterest. The force vector applied is a function of the energy,duration, and location of the pulse of optical illumination. In someembodiments, the pulse of optical illumination can be used to locallyheat the surrounding cell media (i.e. fluidic medium), therebyincreasing the local vapor pressure to create a vapor-fluid interfacethat produces a bubble. The effect upon the surrounding fluidic mediaand/or cell(s) of the heat-induced bubble generation may vary, dependingon the duration and configuration of the microfluidic device and/or thethermal target. Some variety of effects may include:

Cavitation. A short pulse of light may be used to heat the thermaltarget to generate a short-lived bubble. The bubble, upon collapse,creates a cavitating force that may dislodge cells that may be disposednearby. In some embodiments, the short pulse of light is directed at oneor more cells, which may be dislodged by a cavitating force so formed.

Shear Flow. In other embodiments, the bubble may be grown by continuedillumination to create a shear flow of fluid directed towards nearbycell(s) and thereby dislodge the cell(s).

Meniscus Contact. Alternatively, the bubble(s) created by heating thefluidic medium at the site of the thermal target may be directed towardsthe cell(s). As the bubble moves, the meniscus of the bubble may contactthe cell(s) and dislodge them from the surface.

In other embodiments, the bubble can be grown until it isthermodynamically favorable to stabilize and persist in the fluidicmedium. The bubble may then displace the surrounding liquid phase andthereby dislodge the cells.

Convective flow. Without intending to be limited by theory, illuminationof a thermal target which generates heating in the fluidic mediumsurrounding the thermal target may nucleate and propagate bubbles. Thepresence of a persistent bubble having a thermal gradient may produce athermal capillary convection flow (Gibbs-Marangoni effect). TheGibbs-Marangoni effect refers to the flow of liquid along a surfacetension gradient. A liquid may flow from an area of low surface tensionto an area of high surface tension. Because surface tension is decreasedat higher temperature, a temperature gradient on the surface of a bubblecan cause liquid surrounding the bubble to flow in the direction of thegradient (i.e. from the area of high temperature to the area of lowtemperature), and may form a convective flow. For simplicity ofexplanation, the flow created by the temperature gradient on the surfaceof the bubble is herein referred to as a “Marangoni-effect flow.” Thegreater the temperature difference between the hot and cold areas of thesurface, the greater the velocity of the Marangoni-effect flow. Bychanging the optical power, the flow may be modulated. Such a convectiveflow may be used to move cells or mix fluidic media. In someembodiments, a cyclized flow created by heating induced by opticalillumination may be used to dislodge cells within an isolation region ofa sequestration pen. In other embodiments, cyclized flow may be inducedin a localized region of microfluidic devices otherwise devoid offluidic flow, which may be used to mix media in the localized regions.

After dislodging the cell(s), further translocation of the cell(s) maybe accomplished by using any other suitable method of moving cells,including but not limited to fluidic displacement, DEP, gravity, and thelike.

Optical Illumination. The optical illumination can be a coherent lightsource (e.g., a laser) or a non-coherent light source. The coherentlight source may be a laser characterized by a wavelength in the visiblelight spectrum (e.g., a red wavelength, such as 662 nm), or may be alaser characterized by a wavelength in the infrared part of the spectrum(e.g., a near infra-red wavelength, such as 785 nm), or may be a laserhaving any other suitable wavelength. The non-coherent light source maycontain light having wavelengths in the visible range, and/or mayinclude light having wavelengths in the ultraviolet (uv) or infraredrange. The light source may provide structured or unstructured light.Temperature gradients introduced by illumination with a light source,may be modulated by increasing or decreasing the intensity of the lightsource. A structured light source may be modulated in a number of waysto control the properties of the structured light source (e.g. using aDMD to spatially modulate the light source, or using an aperture andobjective to modulate the lights source as described above with respectto FIG. 3B.

Without being bound by theory, the incident optical illumination may betransmitted through a transparent, substantially transparent, and/ortranslucent cover or base of the enclosure microfluidic device. Afterbeing transmitted through the cover or base of the enclosure, theincident illumination can be transmitted to a thermal target, asdescribed below, which is configured to convert the optical illuminationto thermal energy.

Power. Non-coherent light may be projected in a range from about 1milliwatts (mW) to about 1000 milliwatts (mW), but is not limited tothis range. In some embodiments, the power of the non-coherent light,structured or non-structured may be in a range of about 1 milliwatt toabout 500 milliwatts; about 1 milliwatt to about 100 milliwatts; about 1milliwatt to about 50 milliwatts; about 1 milliwatt to about 20milliwatts; about 10 milliwatts to about 500 milliwatts; about 10milliwatts to about 200 milliwatts, about 10 milliwatts to about 100milliwatts; about 50 milliwatts to about 800 milliwatts; about 50milliwatts to about 500 milliwatts; about 50 milliwatts to about 200milliwatts; about 75 milliwatts to about 700 milliwatts; about 75milliwatts to about 400 milliwatts; about 75 milliwatts to about 175milliwatts, or any value therebetween. Depending on the area to whichthe light is focused, and on the duration of illumination, the power ofthe non-coherent light may be less or more than any of the power levelsdescribed above. Coherent light may be projected in a range from about 1milliwatts to about 1000 milliwatts, but is not limited to this range.Depending on the area to which the light is focused, and on the durationof illumination, the power of the coherent light may be less or morethan any of the power levels described above. In some embodiments, thepower of the coherent light may be in a range of 1 milliwatt to about500 milliwatts; about 1 milliwatt to about 100 milliwatts; about 1milliwatt to about 50 milliwatts; about 1 milliwatt to about 20milliwatts; about 10 milliwatts to about 500 milliwatts; about 10milliwatts to about 200 milliwatts, about 10 milliwatts to about 100milliwatts; about 50 milliwatts to about 800 milliwatts; about 50milliwatts to about 500 milliwatts; about 50 milliwatts to about 200milliwatts; about 75 milliwatts to about 700 milliwatts; about 75milliwatts to about 400 milliwatts; about 75 milliwatts to about 175milliwatts, or any value therebetween.

The power of the incident light may be chosen to be different based onthe type of dislodging force desired. For example, if a cyclized flowwhich may incorporate a Marangoni-effect flow is desired, the power ofthe incident light may be selected to be as low as 1 milliwatt andmodulated variously as the cyclized flow is established and/ormaintained. When dislodging micro-objects by use of a cavitating force,shear flow force, or bubble contact force the power may be selected tobe in a higher range, for example from about 10 milliwatts to about 100milliwatts. The power may also be adjusted based on the duration of theillumination desired as well.

Site of illumination. The site of illumination may be selected to be anydiscrete selected region of the microfluidic device, as may be useful.In some embodiments, the discrete selected region of illumination may bea location within a sequestration pen of a microfluidic device. Invarious embodiments, the discrete selected region of illumination islocated within an isolation region of a sequestration pen, which may beconfigured like any sequestration pen described herein, including butnot limited to 124, 126, 128, 130, 224, 226, 228, 266, 502, 504, 506,604, 606, 608, 704, 732, 734, 736, 738, 802, 804, 806, 902, 1002, 1102,1202, 1402, 1502. In various embodiments, the discrete selected regionof illumination may be within a displacement force generation region ofthe sequestration pen, as described more fully below. In otherembodiments, the discrete region of illumination may be within a cyclicculturing pen. When illumination is performed within a cyclic culturingpen, it may be directed at a discrete selected region in a displacementforce generation region, connection region, cell culturing region or atan opening of the cyclic culturing pen to a microfluidic channel. In yetother embodiments, the discrete selected region of illumination may belocated within a microfluidic channel, as described more fully below.

Microfluidic devices The disclosure provides for microfluidic devicesconfigured to be capable of optically driven convection flow generationand/or displacement of micro-objects therein. In one aspect of thedisclosure, a microfluidic device is provided having an enclosure, wherethe enclosure includes a flow region and a sequestration pen, where thesequestration pen may include a connection region and an isolationregion, where the connection region includes a proximal opening to theflow region and a distal opening to the isolation region. Thesequestration pen may include a thermal target in the isolation region.In various embodiments, the sequestration pen further includes adisplacement force generation region, where the isolation regionincludes at least one fluidic connection to the displacement forcegeneration region; and the displacement force generation region furtherincludes a thermal target. In various embodiments, the microfluidicdevice may have at least one sequestration pen which may be configuredas any of sequestration pens 124, 126, 128, 130, 224, 226, 228, 266,502, 504, 506, 604, 606, 608, 704, 732, 734, 736, 738, 802, 804, 806,902, 1002, 1102, 1202, 1402, 1502. In various embodiments, the thermaltarget or the displacement force generation region may be configured toconstrain expansion of a gaseous bubble formed thereupon in onepredominate direction.

In various embodiments, the enclosure of the microfluidic device mayfurther include a cover that defines, in part, the sequestration pen,where the thermal target may be disposed on the cover. In someembodiments, the thermal target may be disposed on an inner surface ofthe cover facing the enclosure. In other embodiments, the enclosure ofthe microfluidic device may further include a microfluidic circuitstructure that defines, in part, the sequestration pen, and the thermaltarget may be disposed on the microfluidic circuit structure. In yetother embodiments, the enclosure of the microfluidic device may furtherinclude a base that defines, in part, the sequestration pen, and thethermal target may be disposed on an inner surface of the base.

In another aspect of the disclosure, a microfluidic device is provided,having an enclosure where the enclosure includes a microfluidic circuitconfigured to contain a fluidic medium, where the microfluidic circuitis configured to accommodate at least one cyclic flow of the fluidicmedium; and a first thermal target disposed on a surface of theenclosure within the microfluidic circuit, wherein the first thermaltarget is configured to produce a first cyclic flow of the fluidicmedium upon optical illumination.

In various embodiments of the microfluidic device comprising amicrofluidic circuit configured to accommodate a cyclized flow, theenclosure of the microfluidic device may further include a microfluidicchannel and a sequestration pen, and further where the sequestration penmay be adjacent to and opens off of the microfluidic channel. In variousembodiments, the sequestration pen may be configured as any ofsequestration pens 124, 126, 128, 130, 224, 226, 228, 266, 502, 504,506, 604, 606, 608, 704, 732, 734, 736, 738, 802, 804, 806, 902, 1002,1102, 1202, 1402, 1502. In other embodiments, the microfluidic devicecomprising a microfluidic circuit configured to accommodate a cyclizedflow, the enclosure of the microfluidic device may further include amicrofluidic channel and a cyclic culturing pen. The cyclic culturingpen may be configured as any of cyclic culturing pens 602, 802, 1302.The cyclic culturing pen may open off of the microfluidic channel andmay further have any other feature or dimension as described herein fora cyclic culturing pen.

In various embodiments of the microfluidic device comprising amicrofluidic circuit configured to accommodate a cyclized flow, thecyclic flow path may include a portion of the channel and at least aportion of the sequestration pen. In other embodiments, the cyclic flowpath may be disposed within the sequestration pen. In some embodiments,the cyclic flow path may include a constricted portion.

In various embodiments of the microfluidic device comprising amicrofluidic circuit configured to accommodate a cyclized flow, themicrofluidic device may include a second thermal target configured toproduce a second cyclic flow of the fluidic medium upon opticalillumination. The second thermal target may be disposed adjacent to thefirst thermal target on the surface of the enclosure. The second thermaltarget may be disposed within the same microfluidic circuit as the firstthermal target. In various embodiments, the first thermal target and thesecond thermal target may be oriented to provide the first cyclic flowand the second cyclic flow of the fluidic medium in opposite directions.

In various embodiments of the microfluidic device comprising amicrofluidic circuit configured to accommodate a cyclized flow, thethermal target is disposed on a surface within the microfluidic channel.In some embodiments, the enclosure of the microfluidic device mayfurther include more than one microfluidic channel, where a firstmicrofluidic channel may be configured to open from a secondmicrofluidic channel at a first location along the second microfluidicchannel and may further be configured to reconnect to the secondmicrofluidic channel at a second location thereby forming themicrofluidic circuit; and the thermal target may be disposed on asurface within the first microfluidic channel. In various embodiments,the at least one sequestration pen may open off of the firstmicrofluidic channel. In various embodiments, the at least onesequestration pen may be configured as any of sequestration pens 124,126, 128, 130, 224, 226, 228, 266, 502, 504, 506, 604, 606, 608, 704,732, 734, 736, 738, 802, 804, 806, 902, 1002, 1102, 1202, 1402, 1502. Insome embodiments, a fluidic resistance of the first channel may beapproximately 10 to 100 times higher than a fluidic resistance of thesecond channel. The second microfluidic channel may have a width that isapproximately 1.5 to 3 times larger than a width of the firstmicrofluidic channel. In some embodiments, the width of the secondmicrofluidic channel is about 100 to 1000 microns. In variousembodiments, the width of the first microfluidic channel may be about 20to 300 microns.

In yet another aspect, a microfluidic device is provided, having anenclosure, where the enclosure includes a microfluidic channel and asequestration pen, and further where the sequestration pen is adjacentto and opens off of the microfluidic channel and a thermal target isdisposed in the channel adjacent to an opening to a sequestration pen,and wherein the thermal target is further configured to direct a flow ofthe fluidic medium into the sequestration pen upon optical illumination.In various embodiments, the at least one sequestration pen may beconfigured as any of sequestration pens 124, 126, 128, 130, 224, 226,228, 266, 502, 504, 506, 604, 606, 608, 704, 732, 734, 736, 738, 802,804, 806, 902, 1002, 1102, 1202, 1402, 1502. In some embodiments, whenthe microfluidic device having a sequestration pen and a thermal targetin the channel has sequestration pens configured as sequestration pens502, 504, 506, 602, 604, 606, 608, 704, 732, 734, 736, 738, 902, thesequestration pens may not have a thermal target within thesequestration pen itself. In some embodiments, the thermal target may bedisposed on a surface within the microfluidic channel.

For any of the microfluidic devices, the enclosure may further include adielectrophoresis configuration. In some embodiments, thedielectrophoresis configuration may be optically actuated.

For any of the microfluidic devices, at least one surface of theenclosure may include a coated surface. In some embodiments, asequestration pen of the microfluidic device may include at least onesurface that is a coated surface. In some embodiments, the coatedsurface may be a covalently modified surface.

Thermal targets. A thermal target is a microfluidic feature of themicrofluidic device which may be a separate feature designed for thispurpose. Alternatively, a thermal target may be a location within themicrofluidic circuit to which optical illumination is applied. Thethermal target is a passive microfluidic feature and does not includeany self-activating resistors or electrical heaters. The passive natureof the thermal targets simplifies fabrication of the microfluidicdevice. For thermal targets including metal or microstructures,fabrication is much less complex than an active thermal target such as aresistor, as is described below. Active thermal targets such asresistors and the like must have fixed electrical connections and arefabricated in fixed positions, unlike the passive thermal targets of thepresent disclosure. when the thermal target is a selected location ofthe microfluidic circuit material or base, with no additional structuralfeature, the flexibility to create forces specifically and selectivelywhere needed is particularly advantageous compared to fixed activethermal targets.

FIGS. 4A-4E illustrate geometries of various thermal targets accordingto some embodiments of the present disclosure. As can be appreciated bythose skilled in the art, any of the characteristics of the thermaltargets depicted in FIGS. 4A-E can be combined to produce thermaltargets with a range of desired functionalities.

FIG. 4A illustrates a thermal target 430 that is square shaped. Theblunt corners and uniform sides of the thermal target 430 of FIG. 4A maybe beneficial in nucleating a substantially uniform bubble. Similarly,the circular thermal target 432 illustrated in FIG. 4B provides a shapeconfigured to generate a uniform localized heat source to nucleate asubstantially uniform bubble.

Conversely, the thermal targets illustrated in FIGS. 4C, 4D, 4E, and 4Ghave asymmetric shapes which may be beneficial in creating bubbles withtemperature gradients. Without intending to be limited by theory,bubbles with temperature gradients may produce a Gibbs-Marangoni effect(also known as thermo-capillary convection), as described above. Theasymmetrical thermal target 434 illustrated in FIG. 4C is characterizedby a teardrop-like shape used to create a bubble with a temperaturegradient that can be used to generate a Marangoni-effect flow. Becausethe wider portion 434 a of the teardrop-like shape has a larger surfacearea than the tapered portion 434 b of the teardrop-like shape, thelarger surface area will generate a higher temperature upon heating byoptical illumination. Consequently, a bubble that is generated using theasymmetrical thermal target 434 in FIG. 4C can have a temperaturegradient in which an area of the bubble that is positioned over thewider portion 434 a of the thermal target will have a higher temperaturethan an area of the bubble that is positioned over the tapered portion434 b of the thermal target.

The temperature gradient may also be modulated by physically separatingdifferently-sized portions of a thermal target. FIG. 4D illustrates athermal target 436 which is comprised of two portions 438, 440 ofdiffering sizes that are physically separated but situated in closeproximity such that they can be heated using the same structured lightsource. The physical separation of the two portions 438, 440 of thethermal target 436 creates a greater temperature differential which canbe used to create a Marangoni-effect flow having greater velocity, andhence, increased force. FIG. 4E illustrates a thermal target 442 thathas been further segregated into three portions 444, 446, 448.

The thermal target 430, 432, 434, 436, 442, 450, 452 illustrated inFIGS. 4A-4G can be created by depositing a contiguous metal shape or anon-contiguous metal shape onto one surface of the microfluidic circuitstructure 108 or cover 110. In some embodiments, the thermal target maybe disposed on an inner surface of the cover 110. The thermal targetfaces towards the interior (chamber/region 202) of the enclosure 102where it may be in contact with fluidic medium. The thermal targets 430,432, 434, 436, 442, 450, 452 can include any type of metal that can beexcited by a light source to produce heat. Suitable metals includechromium, gold, silver, aluminum, indium tin oxide, or any combinationthereof. Other metals (and alloys) are known in the art. The thermaltarget 440 can have a continuous metal surface or can be composed of anon-contiguous shape of metal (e.g. metal shapes such as dots). Variouspatterns can be used to optimize heating and the generation of uniformbubbles.

FIG. 4F illustrates a thermal target 450 comprising a non-contiguousmetal shape. In the embodiment illustrated in FIG. 4F, the shapes aredots. However, any type of metal shape can be used (e.g. squares, lines,cones, squiggles). In addition, a variety of different metal shapes maybe used in the same thermal target.

In some embodiments, non-contiguous metal shapes may be distributed inan increasing concentration in order to enhance the temperature gradientof the thermal target 450. FIG. 4G illustrates a thermal target 452 thathas been patterned with a gradient of metal shapes. By increasing thedensity of distribution of the metal dots in the wider portion 452 a ofthe thermal target 452 and decreasing the density of distribution of themetal dots in the narrower portion 452 b of the thermal target 452, thetemperature gradient of the thermal target 452 may be enhanced.

In some embodiments, the thickness of the metal deposited as acontiguous metal shape or a noncontiguous metal shape(s) in a thermaltarget may be varied in order to enhance the temperature gradient of thethermal target. For example, a thicker deposit of metal may be used togenerate more heat in a larger (or wider) portion of the thermal target,thereby enhancing the temperature gradient. In some embodiments, thethickness of the deposited metal in a thermal target may be in the rangeof about 3 nm to about 50 nm, about 3 nm to about 30 nm, about 5 nm toabout 50 nm, about 5 nm to about 30 nm, about 5 nm to about 25 nm, orany value therebetween.

Microstructures. As described above, in some embodiments, themicrofluidic circuit structure 108 or cover 110 of the enclosure 102 mayhave one or more microstructures introduced thereupon to create asurface topography that may promote bubble nucleation and/or formationfrom heat generated upon optical illumination, and function as a thermaltarget. The microstructure(s) may be a non-contiguous formation Themicrostructure(s) may be a negative feature (e.g., depressions or divotscreated upon the surface of the base or on the surface of a wall. As isknown the art, microstructure(s) such as pillars, dots, cavities ordivots may be patterned into the microfluidic circuit structure 108 orcover 110 in order to create suitable sites for bubble nucleation. FIG.4H is a stylized illustration of a thermal target 454 comprising divotsthat may be used for bubble nucleation. In some embodiments, surfacetopographies may be combined in various ways with metal patterns tocreate an ideal surface for thermal absorption generating subsequentdisplacement forces. The microstructure(s) may have an area in thex-axial and y-axial direction as viewed from above in the range of about50 square microns, 100 square microns; about 200 square microns, about300 square microns, about 500 square microns, or any value therebetween.A microstructure may include only one unit or may be a plurality ofmicrostructures which together have a total area as described. Negativemicrostructure(s) may be formed by focusing a light source (e.g., alaser or a non-coherent light) on patternable microfluidic circuitmaterial that may be disposed on the base or may be part of the walls,where the focused light may pattern the patternable microfluidic circuitmaterial and form the divots or depressions.

Alternatively, the microstructure(s) may be a positive feature, e.g.,rising above the surface of the base or extending from a wall of theenclosure, flow region or sequestration pen (non-limiting examplesincluding pillars or dots (not shown)). The microstructure(s) may haveany conveniently fabricated height within the enclosure. It may have aheight that still permits passage of a micro-object such as a biologicalcell over the microstructure. The height of the microstructure(s) may beabout 5 microns, about 10 microns, about 15 microns, about 20 microns,about 25 microns, about 30 microns, about 35 microns, about 40 microns,or any value therebetween. Each of the plurality of microstructures doesnot have to have the same height but may have a different height fromeach other. The positive microstructure(s) may be formed from the samematerial used to form the microfluidic circuit structure, e.g., PDMS, orany photopatternable silicone, and may be formed during the same processused to fabricate the other elements of the microfluidic circuit, suchas walls, sequestration pens or channels.

In some other embodiments, the positive microstructures may be formedfrom a hydrogel, such as a photo-initiated polymer. The photo-initiatedpolymer may be a synthetic polymer, a modified synthetic polymer, or alight activatable biological polymer. In some embodiments, thebiological polymer may be modified to incorporate moieties providing theability to be light activatable.

In some embodiments, the photo-initiated polymer may include at leastone of a polyethylene glycol, modified polyethylene glycol, polylacticacid (PLA), modified polylactic acid, polyglycolic acid (PGA), modifiedpolyglycolic acid, polyacrylamide (PAM), modified polyacrylamide,poly-N-isopropylacrylamide (PNIPAm), modifiedpoly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinylalcohol, polyacrylic acid (PAA), modified polyacrylic acid,polycaprolactone (PCL), modified polycaprolactone, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin,polysaccharide, modified polysaccharide, or a co-polymer in anycombination. In other embodiments, the polymer may include at least oneof a polyethylene glycol, modified polyethylene glycol, polylactic acid(PLA), modified polylactic acid, polyglycolic acid (PGA), modifiedpolyglycolic acid, polyvinyl alcohol (PVA), modified polyvinyl alcohol,polyacrylic acid (PAA), modified polyacrylic acid, polycaprolactone(PCL), modified polycaprolactone, fibronectin, modified fibronectin,collagen, modified collagen, laminin, modified laminin, polysaccharide,modified polysaccharide, or a co-polymer in any combination. In yetother embodiments, the polymer may include at least one of apolyethylene glycol, modified polyethylene glycol, polylactic acid(PLA), modified polylactic acid, polyglycolic acid (PGA), modifiedpolyglycolic acid, polyvinyl alcohol (PVA), modified polyvinyl alcohol,polyacrylic acid (PAA), modified polyacrylic acid, fibronectin, modifiedfibronectin, collagen, modified collagen, laminin, modified laminin, ora co-polymer in any combination. In some embodiments, thephoto-initiated polymer does not include a silicone polymer. In someembodiments, the photo-initiated polymer may not include a polylacticacid (PLA) or a modified polylactic acid polymer. In other embodiments,the photo-initiated polymer may not include a polyglycolic acid (PGA) ora modified polyglycolic polymer. In some embodiments, thephoto-initiated polymer may not include a polyacrylamide or a modifiedpolyacrylamide polymer. In yet other embodiments, the photo-initiatedpolymer may not include a polyvinyl alcohol (PVA) or a modifiedpolyvinyl alcohol polymer. In some embodiments, the photo-initiatedpolymer may not include a polyacrylic (PAA) or modified PAA polymer. Insome other embodiments, the photo-initiated polymer may not include apolycaprolactone (PCL) or a modified polycaprolactone polymer. In otherembodiments, the photo-initiated polymer may not be formed from afibronectin or a modified fibronectin polymer. In some otherembodiments, the photo-initiated polymer may not be formed from acollagen or a modified collagen polymer. In some other embodiments, thephoto-initiated polymer may not be formed from a laminin or a modifiedlaminin polymer.

Physical and chemical characteristics determining suitability of apolymer for use in the solidified polymer network may include molecularweight, hydrophobicity, solubility, rate of diffusion, viscosity (e.g.,of the medium), excitation and/or emission range (e.g., of fluorescentreagents immobilized therein), known background fluorescence,characteristics influencing polymerization, and pore size of asolidified polymer network. The photo-initiated polymer may be formedupon polymerization of a flowable polymer (e.g., a pre-polymersolution). Briefly, a flowable polymer solution may be flowed into themicrofluidic device and solidified in-situ, prior to use in the methodsdescribed herein. Methods of installing microstructures derived from aphoto-initiated polymer are more fully described in U.S. applicationSer. No. 15/372094 filed on Dec. 7, 2016.

One type of polymer, amongst the many polymers that may be used, ispolyethylene glycol diacrylate (PEGDA). Light initiated polymerizationmay be initiated in the presence of a free radical initiator, such asIgracure® 2959 (BASF), a highly efficient, non-yellowing radical, alphahydroxy ketone photoinitiator, is typically used for initiation atwavelengths in the UV region (e.g., 365 nm), but other initiators may beused. An example of another useful photoinitiator class forpolymerization reactions is the group of lithium acyl phosphinate salts,of which lithium phenyl 2,4,6,-trimethylbenzolylphosphinate hasparticular utility due to its more efficient absorption at longerwavelengths (e.g., 405 nm) than that of the alpha hydroxy ketone class.

Other types of PEG that may be photopolymerized include PEGdimethylacrylate, and/or multiarm PEG (n-PEG) acrylate (n-PEG-Acr).Other polymer classes that may be used include poly vinyl alcohol (PVA),polylactic acid (PLA) polyacrylic acid (PAA), polyacrylamide (PAM),polyglycolic acid (PGA) or polycaprolactone (PCL).

The molecular weight range of the polymer may be varied as required forthe performance of the microstructure(s). A wide range of molecularweights of the flowable polymer may be suitable, depending upon thestructure of the polymer. A useful star type polymer may have Mw (weightaverage molecular weight) in a range from about 500 Da to about 20 kDa(e.g., four arm polymer), or up to about 5 kDa for each arm or for alinear polymer, or any value therebetween. In some embodiments, apolymer having a higher molecular weight range, may be used at lowerconcentrations in the flowable polymer, and still providemicrostructure(s) that may be used in the methods described herein.

Various co-polymer classes may be used, including but not limited to:any of the above listed polymer, or biological polymers such asfibronectin, collagen or laminin. Polysaccharides such as dextran ormodified collagens may be used.

In some embodiments, the microstructure(s) may be or form part of asacrificial feature as used in the methods described herein, and may bedeformed or deteriorated as a result of absorbing optical illuminationand generating thermal effects, which can be used to move micro-objects.

Alternatively, a thermal target may be created in situ by projectingstructured light on the microfluidic circuit structure 108 or cover 110to create patterns of light that have the same geometries as the thermaltarget 450. This approach does not require any special metal depositionor microfluidic circuit material patterning. FIG. 4I provides a stylizedillustration of a thermal target 456 created by projecting a circularpattern of light on the microfluidic circuit structure 108 or cover 110.Projecting patterns of light having particular geometries may also beused in conjunction with thermal targets comprising various geometries,surface topographies, metal patterns and combinations thereof.

In yet other embodiments, optical illumination is focused on portions ofmicrofluidic circuit material of sequestration pen walls, inner surfaceof the base which faces the enclosure of the microfluidic device, orselected point on a microfluidic channel wall, and do not contain any ofthe above described special features of a thermal target. However, thesediscrete selected regions of the microfluidic circuit material ofsequestration pens or walls, or the inner surface of the base may alsobe utilized as thermal targets and may function as a sacrificialfeature.

Size. A thermal target may have a first dimension (e.g., width orx-axial dimension of the microfluidic enclosure) of about 1 mm, 0.9 mm,0.7 mm, 0.5 mm, 0.3 mm, 100 microns, 80 microns, 60 microns, 40 microns,20 microns, about 10 microns, about 5 microns or any value therebetween.The step of illuminating a selected discrete region may further includeilluminating a region having a second dimension (e.g., a y-axialdimension within the microfluidic enclosure) of about 1 mm, 0.9 mm, 0.7mm, 0.5 mm, 0.3 mm, 100 microns, 80 microns, 60 microns, 40 microns, 20microns, about 10 microns, about 5 microns or any value therebetween.The x-axial and y-axial dimensions may be any combination of thedimensions as above. In some embodiments, a thermal target may have anx-axial dimension of about 100 microns and a y-axial dimension of about100 microns. In other non-limiting embodiments, the thermal target mayhave an x-axial dimension of about 5 microns and a y-axial dimension ofabout 5 microns.

Some embodiments of sequestration pens and cyclic culturing pens foroptically driven convection and displacement. As described above, asequestration pen useful for optically driven convection and/ordisplacement of micro-objects may have a displacement force generationregion, which is fluidically connected to the isolation region of thesequestration pen, where micro-objects may be disposed and, optionally,maintained. The displacement force generation region may be connected toa distal portion of the isolation region, opposite to the opening of theisolation region to the connection region. Alternatively, thedisplacement force generation region may be connected to the isolationregion at or adjacent to the opening of the isolation region to theconnection region. In some embodiments, the displacement forcegeneration region may have more than one fluidic connection to theisolation region. (See FIGS. 7A-7F). In some embodiments, thesequestration pen may include a cyclic flow path. The cyclic flow pathmay include the isolation region and the displacement force generationregion. (See FIGS. 6A-6C). In some embodiments, the cyclic flow path mayinclude a constricted portion. (See FIG. 6A).

In some other embodiments, the displacement force generation region mayinclude one or more fluidic connections to the isolation region whichinterrupt the opening of the isolation region to the connection region.In some embodiments, at least one fluidic connection between theisolation region and the displacement force generation region mayinclude a cross sectional dimension configured to prevent passage of amicro-object from the isolation region to the displacement forcegeneration region. Preventing micro-objects from possible entry into thedisplacement force generation region may decrease damage from heat orimpact and may further assist in maintaining the micro-objects withinthe isolation region of the sequestration pen. In some embodiments, theat least one fluidic connection between the isolation region and thedisplacement force generation region includes one or more barriermodules (see FIGS. 7A-7F, 726, 726 a, 726 b, 726 c, 726 d, or 726 e),wherein the one or more barrier modules are configured to preventpassage of a micro-object from the isolation region to the displacementforce generation region. The barrier module(s) may be of any size orshape, and the gap (See FIG. 7A-7F, 728, 728 a, 728 b, 728 c, 728 d, 728e) between a barrier module and its neighbor, or the gap between abarrier module and a wall of the sequestration pen may have a dimensionsuch that a micro-object such as a biological cell may not pass from theisolation region to the displacement force generation region. In someembodiments, a micro-object such as a micro-bead may pass from theisolation region into the displacement force generation region but amicro-object such as a biological cell or an embryo may not pass by thebarrier module(s) into the displacement force generation region. Abarrier module may have a dimension across a sequestration pen that isabout 10%, 20%, 30%, 40%, 50% 60% 70%, about 80%, or any valuetherebetween, of the width of the sequestration pen. The gap between thebarrier module and its neighbor or the gap between a barrier module anda wall may be about 5 microns, 7 microns, 9 microns, 11 microns, 13microns, 15 microns, 17 microns, 20 microns, 25 microns, or about 40microns, depending on the size of the micro-object disposed within theisolation region.

In various embodiments, the at least one fluidic connection between theisolation region and the displacement force generation region may have across sectional dimension configured to prevent fluidic flow from thedisplacement force generation region in the absence of a force generatedtherein, except by diffusion. (See FIGS. 9A-9C.) The dimensions of thedisplacement force generation region may match that of the isolationregion. The displacement force generation region may be a separatecompartment of a sequestration pen. In various embodiments, thedisplacement force generation region may be configured to minimizesecondary flows.

In some embodiments, the sequestration pen may include a second thermaltarget configured to produce a second cyclic flow of the fluidic mediumupon optical illumination. The second thermal target may be disposedwithin the displacement force generation region. The first thermaltarget and the second thermal target may be oriented to provide a firstcyclic flow and a second cyclic flow of the fluidic medium in oppositedirections.

In various embodiments, the displacement force generation region mayinclude a single opening, where the single opening may be the fluidicconnection of the displacement force generation region to the isolationregion. In some embodiments, the fluidic connection of the displacementforce generation region may include a fluidic connector. (See FIG. 5A,fluidic connector 514.) In some embodiments the fluidic connector of thedisplacement force generation region may include at least one curvedportion. (See FIG. 5A, fluidic connector 514.) In some embodiments, theat least one curved portion of the fluidic connector may include a turnof about 60 degrees to about 180 degrees; about 60 degrees to about 120degrees, about 60 degrees to about 90 degrees; about 40 degrees to about180 degrees, about 40 degrees to about 120 degrees, about 40 degrees toabout 90 degrees, or any value therebetween. In other embodiments, thefluidic connector of the displacement force generation region mayinclude at least two curved portions. In some embodiments, each of theat least two curved portions of the fluidic connector may include a turnof about 60 degrees to about 180 degrees; about 60 degrees to about 120degrees, about 60 degrees to about 90 degrees; about 40 degrees to about180 degrees, about 40 degrees to about 120 degrees, about 40 degrees toabout 90 degrees, or any value therebetween. When turns are included inthe fluidic connecter between the displacement force generation regionand the isolation region, the sequestration pen as a whole may have ashape resembling a “U”-like shape, an “N”-like or reverse “N”-likeshape.

In various embodiments, a width of the fluidic connector may be the sameas a width of the isolation region and/or the displacement forcegenerating region. In some embodiments, the fluidic connector mayinclude a cross sectional dimension, in the x-axial and y-axialdimension, configured to prevent passage of a micro-object from theisolation region to the displacement force generation region. In variousembodiments, the height of the fluidic connector, in the z-axialdirection, may also vary, such that a micro-object cannot pass. From theisolation region to the displacement force generation region.

In various embodiments, the thermal target or the displacement forcegeneration region within a sequestration pen may be configured toconstrain expansion of a gaseous bubble formed thereupon in onepredominate direction. For example, the displacement force region may beelongate, and may have the thermal target located at the distal portionof the displacement region such that cavitation force/bubblegrowth/shear flow/convective flow is forced in the direction towards thefluidic connection with the isolation region. This is not a limitingdescription of the configurations of thermal targets or displacementforce generation regions that can constrain expansion of a gaseousbubble, and other configurations are possible.

In various embodiments, the thermal target may be positioned in aportion of the displacement force generation region distal to the leastone fluidic connection to the isolation region. In some embodiments, thedisplacement force generation region may have a width of approximately20-100 microns in an x-axial or y-axial dimension, depending on theorientation of the displacement force generation region within themicrofluidic device. In some embodiments, the displacement forcegeneration region may be configured to minimize secondary flows offluidic media, which maximizes the force directed at the cell(s) in theisolation region.

A cyclic culturing pen may have a connection region and a displacementforce generation region having any combination of features or dimensiondescribed above for a sequestration pen. A cyclic culturing pen mayinclude a culturing region which may have any dimensions or featuresdescribed for an isolation region of a sequestration pen, but differs inthe respect that a cyclic culturing pen is configured to cycle flowthrough the main channel and into the culturing region when activelycycling. In various embodiments, the displacement force generationregion of a cyclic culturing pen may further include an opening to theflow region. One embodiment of a cyclic culturing pen is described morefully below for FIG. 6A.

The configurations of the microfluidic devices of the disclosure andtheir uses may be more fully understood by turning to FIGS. 5A-8D.

FIG. 5A illustrates an example of a sequestration pen 502 opening off ofchannel 522, which is configured to contain a fluidic medium flow 530 inmicrofluidic device 500. Sequestration pen 502 includes a thermal target540 configured to generate bubbles (not shown) used to exportmicro-objects 504 from the sequestration pen 502 according to someembodiments of the present disclosure. The sequestration pen 502includes an isolation region 510 for storing and/or culturingmicro-objects 504 such as cells. The isolation region 510 and thethermal target 540 are physically separated by a displacement forcegeneration region 512 which also includes a fluidic connector 514 whichpermits sufficient space for a bubble to be nucleated and increased involume (referred to herein as “expanding the bubble”) by focusing light(not shown) on the thermal target 540. As the bubble grows by increasingin volume, the expanding bubble generates a force on the fluid in thedisplacement force generation region (512 plus 514), thus creating ashear flow (not shown) of fluid along a path 516. In many embodiments,the thermal target 540 may be positioned at the distal end of thedisplacement force generation region 512 (plus 514) to ensure that thebubble exerts force in a predominate direction as it expands. In someembodiments, either the thermal target or the displacement forcegeneration region is configured to constrain expansion of the bubblesuch that it may expand only in one predominate direction as thermalenergy is continued to be supplied by continued illumination. Thefluidic connector 514 between the isolation region and the displacementforce generation region (512 plus 514) may have a cross sectionaldimension configured to prevent fluidic flow from the displacement forcegeneration region (512 plus 514) in the absence of a force generatedtherein, except by diffusion.

In instances where the micro-objects are biological micro-objects (e.g.biological cells), the displacement force generation region (512 plus514) also serves to physically separate the biological micro-objectsfrom the heat generated by the thermal target 450 upon opticalillumination. In some instances, the expanding bubble also serves toprovide a physical barrier between the micro-objects 504 and the thermaltarget 540. As discussed below, the geometry of the sequestration pen502 may be optimized to maximize the force (and consequent shear flow)from nucleating and expanding a bubble.

The sequestration pen 502 illustrated in FIG. 5A has a shape whichresembles the letter “N” in reverse (i.e., a reverse “N”-like shape),however, other embodiments may use different shapes beneficial ingenerating a shear flow sufficient to displace micro-objects 504 fromthe sequestration pen 502.

In addition, for simplicity, the sequestration pen 502 has beenillustrated without any other features that may be used in practice toprovide other desirable functionalities, such as features used to holdcells in place by force of gravity or traps positioned across from thesequestration pen 502 to collect micro-objects 504 and positionmicro-objects 504 in sequestration pens 502. However, in practice, thesefeatures or any other features described herein for a sequestration penmay be used in conjunction with the sequestration pens 502. Similarly,the sequestration pen 502 illustrated in FIG. 5A is illustrated with asquare thermal target 540 used to generate a uniform bubble. In otherembodiments, other shapes or materials of symmetrical thermal targetsmay be used. In yet other embodiments, thermal target 540 is not presentand optical illumination is directed at microfluidic circuit material506 in the vicinity of thermal target 540 to nucleate a bubble which maybe unstable or stable, which may result in cavitation force, shear flowfluidic force, or bubble contact force to dislodge micro-objects 504.

FIG. 5B illustrates the formation of a bubble 520 and use of a shearflow 542 generated by the growing bubble 520 to displace micro-objects504 from the sequestration pen 502 of FIG. 5A. A light source (notshown) is focused on the thermal target 540 to excite (i.e. heat) thethermal target 540, thereby nucleating a bubble 520 a. By continuing tofocus the light source on the thermal target 540, the bubble 520 a canexpand in volume to produce a successively larger bubble 520 b, 520 c,520 d, 520 e. The increasing size of the bubble 520 creates a force onfluidic media (not shown) in the sequestration pen 502, which in turn,creates a shear flow 542 that displaces micro-objects 504 from theisolation region 510 into the channel 522. Once the micro-objects 504are displaced into the channel 522, they can be manipulated or moved bycontrolling the flow 530 in the channel 522 or may be moved using DEP.

The displacement force generation region 512, which includes its fluidicconnector 514, may be optimized to enhance the shear flow 542 byadjusting the geometry and length of the displacement force generationregion. For example, the length and width of the displacement forcegeneration region between the thermal target 540 and the isolationregion 510 may be optimized to generate shear flow 542. The thermaltarget 540 may be positioned at the distal portion of the sequestrationpen 502 to ensure that the bubble 520 expands in a single direction.Similarly, the distal portion of the displacement force generationregion 512+514 that includes the thermal target 540 may be narrowed inwidth to ensure that the nucleated bubble 520 expands predominately inone direction. Suitable widths for the distal portion of thedisplacement force generation region 512+514 can range fromapproximately 20 to 100 microns. In some embodiments, the fluidicconnector region 514 of the displacement force generation region mayhave the same width as the distal portion of the displacement forcegeneration region 512.

In some embodiments, the displacement force generation region 512+514may optimized to minimize secondary flows that may interfere with theshear flow 542. As discussed below, the displacement force generationregion 512+514 may include a constriction wherein the width of thedisplacement force generation region 512+514 is significantly reduced.Depending on the embodiment, the width of the constriction may beone-half to one-twentieth the width of the displacement force generationregion 512+514. For example, the constriction may have a width rangingfrom approximately 5-50 microns and the displacement force generationregion 512+514 may have a width ranging from approximately 20-100microns. In some embodiments, the displacement force generation region512+514 contains one or more (2, 3, 4 or 5) turns in the fluidicconnector region 514 of the displacement force generation region.

In the instance illustrated in FIG. 5B, the bubble 520 does not comeinto contact with the micro-objects 504; the micro objects 650 are onlysubject to the shear flow 542 generated by growing the bubble. However,in other embodiments of the use of the sequestration pen 502, it may bedesirable or even advantageous to bring the meniscus of the bubble 520into contact with the micro-objects 504 providing a contacting force todisplace the micro-objects 504 from the isolation region 510 and,optionally, export the micro-objects 504 from the sequestration pen 502.The export may be an active export driven by the flow of bubbles or maysimply dislodge the cells such that another force such as DEP may thenexport the cells 504 from the sequestration pen 502. In someembodiments, a length of the displacement force generation region512+514 may be shortened in order to bring the meniscus of the bubble520 into contact with micro-objects 504 within the isolation region 510.Depending on the embodiment, the displacement force generation region512+514 may partially overlap with the isolation region 510, and furthermay not have a fluidic connector 514 that includes any turns.

In other instances, it may be advantageous to nucleate a bubble 520 thatmoves through the sequestration pen 502 to enter the channel 522. Insome instances, the bubble 520 may be used to export micro-objects intothe channel 522. In other instances, the bubble 520 may be used to blockthe channel 522 (e.g. to prevent micro-objects from moving through thechannel 522) and/or redirect a flow 530 of fluidic medium (not shown) inthe channel 522. For example, in microfluidic circuits includingmultiple channels 122 (such as the microfluidic circuit 280 illustratedin FIG. 2F), a bubble 520 may be induced and used as a blockingmechanism to redirect a flow path 106 from a first channel 122 to one ofthe other channels 122.

Any of the modes of dislodging the micro-objects 504, by bubble flow,shear flow, contact with the meniscus of a bubble, or a cavitating forcemay be alternatively be practiced using this configuration.

Other methods and techniques may be used in conjunction withoptically-driven displacement and export of micro-objects from asequestration pen into a channel or other circuit element. For example,the tilting apparatus 190 may be used to tilt (i.e. rotate themicrofluidic circuit on a horizontal axis) or invert the microfluidiccircuit, thereby subjecting the micro-objects to gravitational force,which may be used contemporaneously or as a preliminary step to use ofthe optically-driven methods. Similarly, in some instances, magneticbeads may be used to disrupt or dislodge micro-objects. In theseinstances, magnetic beads may be disposed in a sequestration pen andremoved using magnetic force. The motion of the magnetic beads as theyare removed from the sequestration pen may assist to displace and/ordislodge micro-objects that have become affixed to the sequestrationpen.

FIG. 5C illustrates the use of optical illumination directed at athermal target 541, which is simply a selected discrete region of theinner surface of the displacement force generation region, that can beused to generate a bubble 521 in sequestration pen 502 of microfluidicdevice 500. The selected discrete region forming the thermal target 541does not require any metal deposition nor any special patterning of themicrofluidic circuit material or inner surface of the base. In theembodiment illustrated in FIG. 5C, the light source may be focused on anarea of the sequestration pen 502 in a square pattern of light (notshown). This square pattern of light can heat the microfluidic circuitstructure 108, the inner surface 109, and/or the cover 110 and therebycreating a thermal target 541 at any selected position, that can be usedto nucleate and grow a bubble 521 that generates a shear force 542sufficient to export micro-objects 504 from the sequestration pen 502.Any of the modes of dislodging the micro-objects 504, by bubble flow,shear flow, contact with the meniscus of a bubble, or a cavitating forcemay be alternatively be practiced using this configuration.

FIG. 5D illustrates a sequestration pen 544 of microfluidic device 550,where repeated number elements are defined as above. The sequestrationpen 544 is configured for optically-driven displacement force generationand used to export micro-objects 504 according to some embodiments ofthe present disclosure. The sequestration pen 544 illustrated in FIG. 5Dis characterized by a shape which resembles the letter “U” (i.e., a“U”-like shape), where the isolation region 554 is directly below theproximal opening 534 and the connection region 552. A thermal target 543is situated at the distal end of the sequestration pen 544, within thedisplacement force generation region 556. As in FIG. 5A, thedisplacement force generation region 556 provides sufficient distancebetween the isolation region 554 and the thermal target 543 to allow forthe nucleation of a bubble (not shown) and its use to create acavitating force, a shear force, a bubble that can contact themicro-objects 504, or a stream of bubbles that can dislodgemicro-objects 504 within the isolation region 554, and optionally,displace the micro-objects 504 into the channel 522. The path of thebubbles, shear flow or cavitating forces is illustrated by path 546.

FIG. 5E illustrates a sequestration pen 548 of microfluidic device 560,where repeated numbered elements are defined as above. The sequestrationpen 548 is configured for optically-driven displacement force generationand used to export micro-objects 504 according to some embodiments ofthe present disclosure. The sequestration pen 548 illustrated in FIG. 5Ealso has a reversed “N” shape similar to the sequestration pen 502illustrated in FIGS. 5A and 5B. However, the displacement forcegeneration region in this embodiment includes three sub-regions 566,567, and 568 separating the thermal target 545 and the isolation region564, which is further connected to connection region 562. Thedisplacement force generation includes a distal portion 566 which alsoincludes the thermal target 545; a first fluidic connector 567 which hasthe same dimensions as the distal portion 566 of the displacement forcegenerating region. The displacement force generation region furtherincludes a second constricted fluidic connector 568, which connects tothe isolation region 564, wherein the width (dimension in the x-axialplane as viewing the figure) of the fluidic connector 568 is narrowedrelative to the first fluidic connector 567 and/or the isolation region564. The constricted width of the second fluidic connector 567 serves toprevent bubbles generated at the thermal target 545 from coming intocontact with micro-objects 504 in the isolation region 564. In addition,the constricted width of the second fluidic connector 567 preventsundesirable secondary flows that can disrupt or create aberrant currentsthat interfere with a shear flow or a cavitation force used to displacethe micro-objects 504. Further, the constricted width of the secondfluidic connector 567 prevents passage of micro-objects 504 from theisolation region to the displacement force generation region (566, 567and 568).

FIG. 6A illustrates a cyclic culturing pen 602 and thermal target 622 ofmicrofluidic device 600 used to create a Marangoni-effect flow 680according to one embodiment of the disclosure. The thermal target 622has an asymmetrical teardrop-like shape which when heated using a lightsource 660, creates a bubble 675 with a temperature gradient thatresults in a cyclic Marangoni-effect flow 680. As discussed above withrespect to FIGS. 4C, 4D, 4E, and 4G, a variety of different asymmetricthermal targets may be used to generate a Marangoni-effect flow 680.

In the embodiment illustrated in FIG. 6A, the portion of the thermaltarget 622 that contains a larger surface area is positioned below theportion of the thermal target 622 that contains a smaller surface area.Therefore, the resultant Marangoni-effect flow 680 that can be generatedby temperature gradient on the bubble moves from the bottom portion ofthe bubble 675 to the top portion of the bubble 675 (directed towardsthe proximal opening 634 of the displacement force generation region614, and away from the fluidic connector 616 of the displacement forcegeneration region 614 plus 616), generating a cyclic, in this instancecounter-clockwise, Marangoni-effect flow 680.

In the embodiment illustrated in FIG. 6A, the cyclic culturing pen 602of microfluidic device 600 has a connection region 610, and culturingregion 612 and a displacement force generation region 614 which includesa fluidic connector 616. The cyclic culturing pen 602 may be similar toa sequestration pen, but is configured to cycle flow through the mainchannel when actively cycling. The displacement force generation region614 has a proximal opening 634 to the microfluidic channel 522, and adistal opening 636 from its fluidic connector 616 to the culturingregion 612. Repeated numbered elements are as defined above. When thethermal target 622 is illuminated with light 660, a bubble 675 isnucleated, developing a cyclic flow 680 (Marangoni-effect flow) cyclingthrough both the cyclic culturing pen 602 and the channel 522. Thecyclic flow 680 can be used to mix fluid and/or displace micro-objects(e.g. cells) anywhere in the cyclic culturing pen 602 and adjacentchannel 522. The velocity of the flow, and therefore its displacementforce, may be moderated by moderating the power of illumination, whichmay require as little as 1 milliwatt to initiate. In some instances, thecyclic flow 680 may have at least a portion of a vector of flow in thesame direction as the flow of medium 530 controlled by the media module160 and the media source 178. The cyclic flow 680 may be used to flushmedia in the channel 522 into the culturing region 612. Similarly, thecyclic flow 680 may also be used to displace and export micro-objects inthe cyclic culturing pen 602.

In some other embodiments, a sequestration pen may include othergeometries which may include a circuit for generating a cyclic(Marangoni-effect) flow 680. While the cyclic culturing pen 602illustrated in FIG. 6A includes a circuit that incorporates the mainchannel 522 (referred to herein as an “open-loop” cyclic culturing pen602), other pen geometries may include a circular portion ofmicrofluidic circuit structure within a sequestration pen to create a“closed-loop” sequestration pen (i.e. a circuit that does not includeany portion of the main channel 522).

Depending on the embodiment and the force of the Marangoni-effect flowgenerated by the bubble, open-loop cyclic culturing pens and closed loopsequestration pens can have different sizes and shapes. For examples,the circuit contained within open-loop and closed-loop sequestrationpens may accommodate different volumes of fluid. Similarly, the lengthof the circuit may vary according to the force of the Marangoni-effectflow 680 and the type of thermal target 622 used. As discussed belowwith respect to FIGS. 8C and FIG. 8D, a circuit may comprise an entirechannel.

FIG. 6B illustrates a “closed-loop” sequestration pen 604, ofmicrofluidic deice 620, configured to generate a cyclic Marangoni-effectflow 682. The sequestration pen 604 has a shape that resembles alowercase “b” (i.e. a “b”-like shape). In the sequestration penillustrated in FIG. 6B, the isolation region 632 is positioned directlybelow connection region 630, which has a proximal opening 534 to themicrofluidic channel 522. The closed-loop sequestration pen 604 has acircular channel, however any type of circuit (e.g. a square or apolygonal channel) can be used. The sequestration pen 604 furthercontains an asymmetrical thermal target 624, which is located within adisplacement force generation region 638 having two fluidic connectionsto the isolation region 632, e.g., the two arms of the circular channelleading from and to the isolation region 630. The thermal target 624 maybe heated using a light source 662 to generate a bubble 672 having atemperature gradient, which, in turn, can create a Marangoni-effect flow682 in the closed-loop circular channel. As the circular channel doesnot open to the main channel 522, the Marangoni-effect flow 682 may beused to mix objects or fluidic medium independently from the fluidicmedium in the main channel 522.

FIG. 6C illustrates a sequestration pen 606 of microfluidic device 640configured to generate a Marangoni-effect flow 684. The sequestrationpen 606 includes an isolation region 664 located directly below aconnection region 642, which has a proximal opening 534 to the channel530. The sequestration pen 606 also surrounds a portion of microfluidiccircuit material providing a closed-loop circular channel having twofluidic connections to the isolation region 644 from the displacementforce generation region 646. The asymmetrical thermal target 626, withinthe displacement force generation region 646, can be heating using alight source 664 to generate a bubble 674 with a temperature gradientthat generates a Marangoni-effect flow 684.

FIG. 6D illustrates a sequestration pen 608 of microfluidic device 670,which is configured to generate Marangoni-effect flows in alternatedirections. As for sequestration pen 606 of microfluidic device 640,sequestration pen 608 has an isolation region 654, a displacement forcegeneration region 656 which is connected to the isolation region via twofluidic connections (arms of the cyclic channel), and a connectionregion 652 having a proximal opening 534 to the channel 522. Thesequestration pen 608 has two thermal targets 628, 629 configured togenerate Marangoni-effect flows (not shown) in alternate directions.Alternating the direction of Marangoni-effect flow can be used to createan agitating motion on micro-objects or fluidic media in thesequestration pen 608, which may serve to provide an enhanced effect inmixing and dislodging micro-objects and media.

FIGS. 7A-7F illustrate other embodiments of sequestration pens usefulfor optically-driven convective flow and micro-object displacement. Ineach of the embodiments shown in FIGS. 7A to 7F, a barrier creates aphysical separation of the displacement force generation region from theisolation region of the sequestration pen. The gap between a barrier,which may be a single barrier module or may be a plurality of barriermodules, and a wall of the sequestration pen provides a fluidicconnection between the two regions but is configured to prevent passageof a micro-object from the isolation region to the displacement forcegeneration region. Similarly, the gap between a barrier module and anadjacent barrier module provides a fluidic connection between the tworegions but is configured to prevent passage of a micro-object from theisolation region to the displacement force generation region. In eachcase, the barrier module(s) also may be configured to prevent damage tothe micro-objects from direct impact of forces generated by theoptically-driven convective and displacement forces, and may also assistin channeling shear flow, cavitation force, or bubble force to moreeffectively dislodge micro-objects within the isolation region. Numberedelements having the same numbers are equivalent.

In FIG. 7A, a sequestration pen 704 of microfluidic device 700, opensoff of microfluidic channel 722, which is configured to contain a flowof fluidic medium 706. The microfluidic channel 706 and the walls of thesequestration pen are fabricated from microfluidic circuit material 716.The sequestration pen 704 has a connection region 714 which has aproximal opening 710 to the microfluidic channel 722. The connectionregion is fluidically connected to an isolation region 712 wheremicro-objects 702 may be disposed and/or maintained. The isolationregion 712 is further connected to displacement force generation region718, which includes a thermal target 724, which may be any thermaltarget as described herein. Sequestration pen 704 also includes a singlebarrier module 726 which forms a boundary between the isolation region714 and the displacement force generation region 718. There are twofluidic connections between the isolation region 714 and thedisplacement force generation region 718, which are the gaps 728,between the barrier 728 and the wall of the sequestration pen 704. Thesequestration pen 704 may be used in the methods of optically-drivenconvective flow and micro-object displacement. In some embodiments, thethermal target may be illuminated by a light source, which may be acoherent or a noncoherent light source, and may be structured orunstructured. In some embodiments, the thermal target is an additionalfeature within the sequestration pen 704 and may be fabricated frommetal, patternable microfluidic circuit material, or photoinitiatedhydrogel polymer, which may be deposited on the cover above thesequestration pen 704 or may be deposited on the surface of the base708. In some embodiments, the thermal target fabricated for this purposeis a sacrificial feature. In other embodiments, the discrete selectedregion that is illuminated is a selected location on the upper surface708 or the microfluidic circuit material 726 of the walls of thedisplacement force generation region. Typically, when the upper surface708 or microfluidic circuit material 716 is illuminated, it behaves as asacrificial feature, generating heat but also being destroyed by theprocess. The duration of illumination may determine what kinds ofdisplacement force are being generated. A short pulse as describedherein, in one non-limiting example of a range of about 10 microsec toabout 200 microsec, may create a cavitation force for displacement. Alonger duration of illumination, in one non-limiting example of about1000 millisec to about 2000 millisec, may provide one of a bubblecontact force, a bubble flow force, bubble meniscus force, or a shearflow force which can dislodge one or more micro-objects within theisolation region. 712. The force dislodging the one or moremicro-objects may be sufficient to displace the cells entirely from theisolation region into the microfluidic channel 722 or may be sufficientto dislodge the micro-objects from the surface of the isolation region712, but not enough to export the cells from the sequestration pen 704.

FIG. 7B shows another arrangement where sequestration pen 730 ofmicrofluidic device 720 has a plurality of barrier modules 726 aseparating the displacement force generation region 718 from theisolation region 712. The displacement force generation region 718 isfluidically connected to the isolation region 712 via a plurality offluidic connections, gaps 728 a.

FIG. 7C represents another variation of a sequestration pen 732 ofmicrofluidic device 740, where a plurality of elongate barrier modules726 b separate the displacement force generation region 718 from theisolation region 712. The displacement force generation region 718 isfluidically connected to the isolation region 712 via a plurality offluidic connections, gaps 728 b.

FIG. 7D is yet another variation, having a sequestration pen 734 ofmicrofluidic device 750. A single barrier module 726 c, has an arc inits configuration which may protect the micro-objects from directimpact. The displacement force generation region 718 is fluidicallyconnected to the isolation region 712 via two fluidic connections, gaps728 c.

FIG. 7E is a further variation, having a sequestration pen 736 ofmicrofluidic device 760. A single barrier module 726 d, has a narrowingprotrusion in its configuration which may help direct the displacingforces more effectively to displace the micro-objects. The displacementforce generation region 718 is fluidically connected to the isolationregion 712 via two fluidic connections, gaps 728 d.

FIG. 7F represents another variation of a sequestration pen 738 ofmicrofluidic device 780, where a plurality of barrier modules 726 edefine the displacement force generation region 718 as a circular regionsurrounding the thermal target 724 and separate the region 718 from theisolation region 712. The displacement force generation region 718 isfluidically connected to the isolation region 712 via a plurality offluidic connections, gaps 728 e.

Sequestration pens 730, 732, 734, 736 and/or 738 may be fabricated inany similar manner to the fabrication of sequestration pen 704, and maybe employed in any method of optically-driven convective flow and/ormicro-object displacement as the methods described for sequestration pen704.

FIG. 8A illustrates a microfluidic device comprising a series ofsequestration pens 802, 804, 806 with asymmetrical thermal targets 840,842, 844 situated within the channel and extending into thesequestration pens 802, 804, 806. A thermal target 842 may be heatedusing a light source 860 to nucleate a bubble 870 with a temperaturegradient which, in turn, creates a Marangoni-effect flow 880 which canbe used to disrupt or displace micro-objects 504 in the sequestrationpen 804 into the channel 822, creating a cyclized flow within thesequestration pen 804. The Marangoni-effect flow 880 can also be used tointroduce fluidic media from the channel 822 into the sequestration pen804. Although the thermal targets 840, 842, 844 illustrated in FIG. 8Aare positioned above the sequestration pens 802, 804, 806 and can beused to introduce fluidic media into unswept portions of thesequestration pens 802, 804, 806, thermal targets configured to producea Marangoni-effect flow 880 may be positioned anywhere in themicrofluidic circuit where it is beneficial to introduce fluid from aswept region to an unswept region. The velocity, and resultant force ofthe cyclized flow may be modulated by increasing or decreasing the powerof the illumination, thereby speeding or slowing the velocity of thecyclized flow.

FIG. 8B illustrates a microfluidic device 810 having an asymmetricalthermal target 846 placed at the terminus of a channel 822. When thethermal target 846 is used to generate a bubble 872 with a temperaturegradient, the resultant Marangoni-effect flow 882 can be used in placeof, or in combination with, the flow path 830 in the channel to moveobjects within the channel 822.

FIG. 8C illustrates another microfluidic device 812 which includes amain channel 824 and ten side channels 826 a-j extending perpendicularlyfrom the main channel 824. Each of the ten side channels connects toanother side channel to form a microfluidic circuit with the mainchannel 824. Specifically, 826 a connects with 826 b, 826 c connectswith 826 d, 826 e connects with 826 f, 826 g connects with 826 h, and826 i connects with 826 j to form circuits. Each of the fivemicrofluidic circuits so formed includes an asymmetrical thermal target848 a-e configured to generate a bubble that causes a Marangoni effectflow. In the embodiment illustrated in FIG. 8C, there is a much lowerfluidic resistance in the main channel 824 than the side channels 826a-e. Due to the difference in fluidic resistance between the mainchannel 824 and the side channels 826 a-e, a flow of fluidic mediaintroduced into the main channel 824 will not enter the side channels826 a-e. In other words, the side channels 826 a-e are unswept regionsof the microfluidic device 812, in the absence of the cyclized flowinduced by optical illumination.

Depending on the embodiment, the ratio of fluidic resistance between themain channel 824 and the side channels can vary. In most embodiments,the fluidic resistance in the main channel 824 at the point at which itbranches into the side channel 826 will be 10 to 100 times lower thanthe fluidic resistance of the side channel 826. Since fluidic resistanceis proportional to the length of the channel and inversely proportionalto the width of a channel, the side channels will typically be longerand narrower than the main channel to achieve the optimal ratio offluidic resistance between the main channel and the side channels. Insome embodiments, the main channel can be 1.5 to 3 times wider than theside channels. For example, the main channel can have a width rangingfrom 100 microns to 1000 microns and the side channel can have a widthranging from 20 microns to 300 microns.

However, as illustrated in FIG. 8D, when bubbles 874 a and 874 b aregenerated using asymmetrical thermal targets 848 a and 848 c, theresultant Marangoni-effect flows generated by the bubbles 874 a and 874b may be used in conjunction with the flow in the main channel 824 toselectively introduce fluidic media from the main channel 824 to theside channels 826 a, 826 b, 826 e, 826 f. In this way, theMarangoni-effect flows may be used to selectively introduce mediacontaining analytes, reagents and or micro-objects (e.g. beads) intochannels of interest to perform assays or culture micro-objects.

Kits. Kits are provided for optically driven devices and methods ofconvective flow and/or micro-object displacement, which includes anymicrofluidic device as described herein, where the microfluidic devicemay include any features described herein in any combination, andreagents for providing a coated surface. The microfluidic device may beselected from any of microfluidic devices 100, 200, 230, 250, 280, 290,500, 550, 560, 600, 620, 640, 670, 700, 720, 720, 750, 760, 780, 808,810, 812, 900, 1000, 1100, 1200, 1300, 1400, 1500. The reagents for acoated surface may be any reagent as described herein for that purpose.The reagent for providing a coated surface may include a reagent thatprovides a covalently linked surface.

In some embodiments of the kit, one or more fluidic media may beprovided. In other embodiments, the kit may include a photoactivatablehydrogel, which may be already formulated as a flowable polymer or maybe a dry powder or lyophilized product. In some embodiments, the kit mayfurther include a photoinitiator. The components of the kit may beprovided in one or more containers.

Method of fabricating sequestration pens having thermal targets. Thermaltargets may be fabricated during the routine manufacture of themicrofluidic devices. Metal targets may be deposited on the cover of themicrofluidic devices during the same operation that adds metalliccontacts for electrical connections and the like. Thermal targetsfabricated from microfluidic circuit material may be included in themask during soft lithography. Thermal targets installed in this fashionmay include sacrificial targets. Surface topographies for bubblegeneration may be patterned into the microfluidic circuit structure 108or cover 110 during fabrication or may be patterned in situ using alight source (not shown). In embodiments where a patternable material isused, a structured light source may be used. Hydrogel thermal targetsmay be installed after the microfluidic device has been fabricated butbefore use in the methods of the disclosure.

Methods of dislodging one or more micro-objects and/or mixing fluidicmedium. Accordingly a method is provided for dislodging one or moremicro-objects (e.g., a biological micro-object such as a cell) from asurface within a microfluidic device, illuminating a selected discreteregion containing or adjacent to one or more micro-objects disposedwithin a fluidic medium in an enclosure of the microfluidic device,wherein the enclosure comprises a microfluidic circuit including a flowregion and a substrate; maintaining the illumination of the selecteddiscrete region of a first period of time sufficient to generate adislodging force, dislodging the one or more micro-objects from thesurface.

The method may include a step of maintaining the one or moremicro-objects within the fluidic medium in the enclosure for a secondperiod of time before performing the step of illuminating the selecteddiscrete region. During maintenance of the cells within the fluidicmedium within the enclosure, some types of cells may become attached toone or more internal surfaces of the enclosure of the microfluidicdevice. The attachment may be non-specific or a specific interactionbetween the cells and the one or more surfaces. A specific interactionmay include a covalent or non-covalent attachment of a surface moietysuch as a carboxylic acid of a cell with an oxide moiety of the surface,which may form hydrogen bonds or ester bonds upon association. Theattachment of the one or more cells may be direct or non-direct to theone or more surfaces. A non-limiting example of a direct attachmentwould be an interaction of a portion of the one or more cells with anoxide moiety of a surface having oxide moieties upon the surface. Anon-limiting example of an indirect attachment of the one or more cellswith a surface may include an interaction between a portion (includingbut not limited to a moiety on the surface of a cell) of the cell withan intervening substance or material that has itself become associatedwith the surface, such as, but not limited to, surface fouling proteinsproduced by other cells present within the enclosure. These arenon-limiting examples of the types of attachment possible between cellsand the surfaces upon which the cells are maintained. Attachment of anykind may decrease the portability of one or more cells.

In various embodiments, the enclosure of the microfluidic device mayfurther include at least one sequestration pen. In some embodiments, themicrofluidic device may include a plurality of sequestration pens. Eachof the plurality of sequestration pens may have a proximal opening tothe flow region. In some embodiments, the flow region may include amicrofluidic channel.

In some embodiments, the surface upon which the one or moremicro-objects are maintained may be a surface of the substrate. Thesurface of the substrate upon which the one or more micro-objects aremaintained may be a surface of the substrate within the at least onesequestration pen.

In various embodiments, the step of illuminating a selected discreteregion may include illuminating a region having a first dimension (e.g.,width or x-axial dimension of the microfluidic enclosure) of about 1 mm,0.9 mm, 0.7 mm, 0.5 mm, 0.3 mm, 100 microns, 80 microns, 60 microns, 40microns, 20 microns, about 10 microns, about 5 microns or any valuetherebetween. The step of illuminating a selected discrete region mayfurther include illuminating a region having a second dimension (e.g., aheight or y-axial dimension within the microfluidic enclosure) of about1 mm, 0.9 mm, 0.7 mm, 0.5 mm, 0.3 mm, 100 microns, 80 microns, 60microns, 40 microns, 20 microns, about 10 microns, about 5 microns orany value therebetween. The x-axial and y-axial dimensions may be anycombination of the dimensions as above. The selected discrete region ofillumination may have an area of about 200 square microns, about 150square microns, about 100 square microns, about 80 square microns, about70 square microns, about 50 square microns, about 25 square microns,about 10 square microns, or any value therebetween.

Period of illumination. The step of illumination may be performed usingany light source as described herein, and may be a coherent or anon-coherent light. The light may be structured or unstructured light.For simplicity, the following description refers to laser illumination,but the invention is not so limited.

In various embodiments, the step of illuminating the selected discreteregion may include illuminating the selected discrete region with alaser. The laser may irradiate with light having a wavelength in theregion of about 450 nm to about 800 nm. The laser may have a current ofabout 0.5 amps, 0.7 amps, 0.9 amps, 1.1 amps, 1.4 amps, 1.6 amps, 1.6amps, 2.0 amps, 2.2 amps, 2.5 amps, 2.7 amps, 3.0 amps, or any valuetherebetween.

The laser illumination may have incident power in the range of about 1mW to about 1000 mW, about 100 mW to about 1000 mW, about 100 mW toabout 800 mW, about 100 mW to about 600 mW, about 100 mW to about 500mW, or any range or individual value therebetween.

In various embodiments, the step of illuminating the selected discreteregion with laser illumination may be performed for a period of time ina range of about 10 microsec to about 8000 millisec, and may be anyvalue therebetween. In some other embodiments, the step of illuminatingthe selected discrete region may be performed for a period of time inthe range of about 100 millisec to about 3 minutes.

In various embodiments, the laser illumination may be directed to theselected discrete region for about 50 millisec, 75 millisec, 100millisec, 150 millisec, 250 millisec, 500 millisec, 750 millisec, orabout 1000 millisec. In various embodiments, the laser illumination maybe directed to the selected discrete region for a period of time in arange of about 50 millisec to about 2000 millisec; about 50 millisec toabout 1000 millisec; about 50 millisec to about 500 millisec; about 50millisec to about 300 millisec; 100 millisec to about 1000 millisec;about 200 millisec to about 1000 millisec; about 200 millisec to about700 millisec; about 300 millisec to about 600 millisec; or any valuetherebetween of any of the ranges. In other embodiments, the laserillumination may be directed to the selected discrete region for aperiod of time in a range of about 1 millisec to about 200 millisec;about 1 millisec to about 150 millisec; about 1 millisec to about 100millisec; about 1 millisec to about 50 millisec; about 1 millisec toabout 30 millisec; about 25 millisec to about 200 millisec; about 25millisec to about 100 millisec; about 25 millisec to about 75 millisec;about 50 millisec to about 200 millisec; about 50 millisec to about 125millisec; about 50 millisec to about 90 millisec; or may be any valuetherebetween of any of the ranges. A period of illumination selected inone of these ranges may be sufficient to optically drive generation of abubble which can contact a micro-object and thereby dislodge it.

In various other embodiments, the laser illumination may be directed tothe selected discrete region for a period of time in a range of about500 millisec to about 3000 millisec; about 1000 millisec to about 2700millisec; about 1000 millisec to about 2500 millisec; about 1000millisec to about 2000 millisec; about 1000 millisec to about 1500millisec; about 1300 millisec to about 3000 millisec; about 1300millisec to about 2700 millisec; about 1300 millisec to about 2300millisec; about 1300 millisec to about 2000 millisec; about 1300millisec to about 1700 millisec; about 1500 millisec to about 3000millisec; about 1500 millisec to about 2600 millisec; about 1500millisec to about 2300 millisec; about 1500 millisec to about 2000millisec; about 1700 millisec to about 3000 millisec; about 1700millisec to about 2600 millisec; about 1700 millisec to about 2000millisec; or any value therebetween. A period of illumination chosen inone of these ranges may be suitable for generating optically-drivenshear flow or bubble flow contact force.

In yet other embodiments, the step of illuminating the selected discreteregion may be performed for about 10 microsec to about 200 millisec;about 10 microsec to about 100 millisec; about 10 microsec to about 1millisec; about 10 microsec to about 1 millisec; about 10 microsec toabout 500 microsec; about 50 microsec to about 1 millisec; about 50microsec to about 500 microsec; about 50 microsec to about 300 microsec;about 1 millisec to about 200 millisec; about 1 millisec to about 150millisec; about 1 millisec to about 100 millisec; about 1 millisec toabout 50 millisec; about 1 millisec to about 30 millisec; about 25millisec to about 200 millisec; about 25 millisec to about 100 millisec;about 25 millisec to about 75 millisec; about 50 millisec to about 200millisec; about 50 millisec to about 125 millisec; about 50 millisec toabout 90 millisec; or may be any value therebetween of any of theranges. A period of illumination in one such range of illumination maybe sufficient to create a cavitating force within a discrete selectregion containing or adjacent to micro-objects, thereby dislodging oneor more of the micro-objects. In some embodiments, the period of time ofillumination may be in a range from about 10 microsec to about 500microsec or from about 10 microsec to about 100 millisec.

In some other embodiments, the step of illuminating the selecteddiscrete region may be performed for about 100 millisec to about 3minutes; about 100 millisec to about 2 minutes; about 100 millisec toabout 1 minute; about 100 millisec to about 10,000 millisec; about 100millisec to about 5,000 millisec; about 100 millisec to about 1000millisec; about 500 millisec to about 3 minutes; about 500 millisec toabout 1 minute; about 500 millisec to about 10,000 millisec; about 500millisec to about 3,000 millisec; or any value therebetween. A period ofillumination in a range selected from one of these ranges may besufficient to create a cyclized flow (Marangoni effect) for mixingfluidic media and/or micro-objects. The period of illumination forcyclized flow may be lengthened or shortened, depending on the powerused to illuminate the discrete selected region.

These ranges are exemplary only and are not intended to limit thedisclosure. Periods of illumination outside of the ranges described foreach type of convective flow or displacement force may be identified andused while still remaining within the scope of the disclosure.

In some embodiments, the step of illuminating a discrete region includesdirecting laser illumination at a selected discrete region containing atleast one of the one or more micro-objects. This may be performedanywhere within the enclosure of the microfluidic device. In someembodiments, the discrete region that is illuminated may be within asequestration pen, and may further be a surface of the substrate withinthe sequestration pen. When illuminating at least one micro-object ofthe one or more micro-objects within a sequestration pen, the selecteddiscrete region may be selected to be at a location distal to a proximalopening of the sequestration pen (e.g., at the bottom or base of thesequestration pen) to the flow region or at a central location withinthe at least one sequestration pen).

The laser illumination may directly cause a dislodging force upon atleast one of the one or more micro-objects. Without being bound bytheory, the illumination may also or alternatively, heat a portion ofthe fluidic medium around the one or more micro-objects and create acavitating dislodging force which can dislodge at least some of the oneor more micro-objects.

In other embodiments of the method, the selected discrete region to beilluminated may be adjacent to the one or more micro-objects. Theselected discrete region may be located about 1 mm, 0.9 mm, 0.7 mm, 0.5mm, 0.3 mm, 100 microns, 80 microns, 60 microns, 40 microns, 20 microns,about 10 microns, about 5 microns or any value therebetween, away fromthe one or more micro-objects to be dislodged. The laser illuminationadjacent to the one or more micro-objects may be performed anywherewithin the enclosure of the microfluidic device. In some embodiments,the step of illuminating the selected discrete region adjacent to theone or more micro-objects may be performed on the substrate; onmicrofluidic circuit material of a wall; or a thermal target, which maybe any thermal target described herein which may further be asacrificial feature. In some embodiments, when the one or moremicro-objects are maintained within a sequestration pen, the step ofilluminating the substrate may be performed on a selected discreteregion on the substrate near a proximal opening of the at least onesequestration pen to the flow region. In other embodiments, when the oneor more micro-objects are maintained within the at least onesequestration pen, the step of illuminating the selected discrete regionmay include illuminating a selected discrete region of microfluidiccircuit material of the at least one sequestration pen. In yet otherembodiments, when the one or more micro-objects are maintained withinthe at least one sequestration pen, the step of illuminating theselected discrete region may include illuminating a sacrificial featuredisposed within the at least one sequestration pen.

A sacrificial feature may be made of any suitable material that mayabsorb energy from the laser illumination, which can include a metal pador microfluidic circuit material (e.g., the same or similar material asthat of the sequestration pen walls and the walls of the flow region(e.g., microfluidic channel). In some embodiments, the sacrificialfeature may include the upper surface of the substrate (which may or maynot include additional coatings or covalently modified surface layers)or any other material that may be included within the microfluidicdevice.

The illumination (e.g., including but not limited to laser illumination)may directly cause a dislodging force upon at least one of the one ormore micro-objects. Without being bound by theory, the illumination mayalso or alternatively, heat a portion of the fluidic medium around theone or more micro-objects and create a cavitating dislodging force whichcan dislodge the one or more micro-objects.

The laser illumination may directly cause a dislodging force upon atleast one of the one or more micro-objects. Without being bound bytheory, the step of illuminating the selected region adjacent to the oneor more micro-objects may also or alternatively, heat a first portion ofthe fluidic medium; and, create a persistent bubble displacing a secondportion of the fluidic medium surrounding the one or more micro-objects,thereby dislodging the one or more micro-objects. The step of displacinga second portion of the fluidic medium may further include creating acyclic fluidic flow of the fluidic medium during the first period ofillumination. In other embodiments, the method may further includeheating a first portion of the fluidic medium; and creating one or moregaseous bubbles, thereby creating a shear flow of the fluidic mediumtowards the one or more micro-objects. In yet other embodiments, themethod may further include heating a first portion of the fluidicmedium; creating a plurality of gaseous bubbles configured to streamtowards the one or more micro-objects; and contacting the one or moremicro-objects with a meniscus of at least one gaseous bubble of theplurality of gaseous bubbles.

The laser illumination may be directed anywhere as described above.Alternatively, when the one or more micro-objects are maintained withina sequestration pen within the enclosure, the selected discrete mayinclude at least a part of a wall forming a distal end of thesequestration pen, wherein the wall is positioned opposite to a proximalopening to the flow region. Illumination at the base of thesequestration pen may prevent damage to the one or more micro-objects.

Alternatively, when the one or more micro-objects are disposed within asequestration pen within the enclosure, the selected discrete region maybe located in a displacement force generation region of thesequestration pen. In some embodiments, the one or more micro-objectsmay be disposed within an isolation region of the sequestration pen andthe displacement force generation region is fluidically connected to theisolation region.

In various embodiments of the method for dislodging one or moremicro-objects within a microfluidic device, the method may furtherinclude a step of exporting the one or more micro-objects from the atleast one sequestration pen. The step of exporting the one or moremicro-objects from the at least one sequestration pen may include movingthe one or more micro-objects with dielectrophoresis force.

In various embodiments of the method for dislodging one or moremicro-objects within a microfluidic device, the method may furtherinclude a step of exporting the one or more micro-objects from the flowregion of the enclosure of the microfluidic device. The step ofexporting the one or more micro-objects from the flow region may includeusing gravity, fluidic flow, dielectrophoresis forces, or anycombination thereof.

In certain embodiments, the disclosure further provides machine-readablestorage devices for storing non-transitory machine readable instructionsfor carrying out the foregoing methods. The machine-readableinstructions can further control the imaging device used to obtain theimages.

In another aspect, a method is provided for mixing fluidic media, and/ormicro-objects contained therein, within an enclosure of a microfluidicdevice, the method including the steps of: focusing a light source on athermal target disposed on a surface of the enclosure within amicrofluidic circuit including at least one fluidic medium and/ormicro-objects, thereby heating a first portion of the at least onefluidic medium; and inducing a cyclic flow of the at least one fluidicmedium within the microfluidic circuit thereby mixing the fluidic mediaand/or micro-objects disposed therein. In some embodiments of themethod, wherein the thermal target is disposed within a firstmicrofluidic channel, which is configured to branch off of a secondfluidic channel at a first location and is also configured to rejoin thesecond fluidic channel at a second location, wherein the thermal targetis disposed on the surface therebetween.

Experimental

System and Microfluidic device: Manufactured by Berkeley Lights, Inc.The system included at least a flow controller, temperature controller,fluidic medium conditioning and pump component, light source for lightactivated DEP configurations, laser, mounting stage for a BerkeleyLights, Inc. OptoFluidic™ microfluidic device, and a camera. TheBerkeley Lights, Inc. Optofluidic™ microfluidic device included NanoPen™chambers having a volume of about 7×10⁵ cubic microns.

Materials: Cells, unless otherwise noted, were OKT3 cells, a murinemyeloma hybridoma cell line, which were obtained from the ATCC (ATCC®Cat. #CRL-8001™). The cells were provided as a suspension cell line.Cultures were maintained by seeding about 1×10⁵ to about 2×10⁵ viablecells/mL and incubating at 37° C., using 5% carbon dioxide in air as thegaseous environment. Cells were split every 2-3 days. OKT3 cell numberand viability were counted and cell density was adjusted to 5×10⁵/ml forloading to the microfluidic device.

Culture medium: 500 ml Iscove's Modified Dulbecco's Medium (ATCC®Catalog No. 30-2005), 200 ml Fetal Bovine Serum (ATCC® Cat. #30-2020)and 1 ml penicillin-streptomycin (Life Technologies® Cat. #15140-122)were combined to make the culture medium. The complete medium wasfiltered through a 0.22 micron filter and stored away from light at 4°C. until use.

Priming procedure: 250 microliters of 100% carbon dioxide was flowed inat a rate of 12 microliters/sec. This was followed by 250 microliters ofPBS containing 0.1% Pluronic® F27 (Life Technologies® Cat#P6866), flowedin at 12 microliters/sec. The final step of priming included 250microliters of PBS, flowed in at 12 microliters/sec. Introduction of theculture medium follows.

Perfusion regime (during cell culturing on chip: The perfusion methodwas either of the following two methods:

-   -   1. Perfuse at 0.01 microliters/sec for 2 h; perfuse at 2        microliters/sec for 64 sec; and repeat.    -   2. Perfuse at 0.02 microliters/sec for 100 sec; stop flow 500        sec; perfuse at 2 microliters/sec for 64 sec; and repeat.

Optical system. For Examples 1 and 2, the optical system included a 785nm laser, Olympus microscope Prosilica camera and an epifluorescencelight train with specialized collimation optics for the laser.

Example 1

Optical illumination of a metal thermal target. FIGS. 9A-D depict theuse of a thermal target to generate bubbles used to export cells from asequestration pen. The sequestration pen 902 of microfluidic device 900shown in FIGS. 9A-9C had a reverse “N”-like geometry similar to thesequestration pen illustrated in FIG. 5A, and includes a connectionregion 906, isolation region 908 where human hybridoma cells 904 werecultured; and a displacement force generation region 910 (labeled inFIG. 9C) including a tripartite fluidic connector 909 having a proximalnarrowed segment 912, connecting to the isolation region, and a distalnarrowed segment 914 connecting to the reservoir region 913 of thedisplacement force generation region 910. The proximal narrowed segment912 of the displacement force generation region 910 has a width that issmaller than a diameter of the cell, and prevents any cells frommigrating from the isolation region 908 to the displacement forcegeneration region 910, in particular segregating the cells from thereservoir region 913, where the optical illumination is focused andheating is most intense. The displacement force generation regionfurther includes a thermal target 916 in the reservoir region 913, acontiguous metal shape formed from gold (Au) that had been depositedonto the inner surface of the cover of the microfluidic device. FIG. 9Ashowed the sequestration pen with a plurality of cells after culturingin the isolation region for three days prior to optical illumination.

The thermal target was heated for 5-10 seconds using a 785 nm laser witha current ranging from 0.8-1.0 Amperes. FIG. 9B is a photograph taken ata timepoint within the illumination period, where a bubble (not shown)had formed at the thermal target 916 which displaced the cells from theisolation region 908 and exported the cells 904 into the connectionregion 906 as well as the microfluidic channel 922 proximate to thesequestration pen 902. FIG. 9C depicts the sequestration pen at a timepoint after the cells have been exported. Exported cells 904 weretransferred to a standard wellplate, and plated individually. Afterthree days of culturing within the wellplate, the cells 904 demonstratedviability by expanding into a larger cell population (FIG. 9D.)

Example 2

Optical illumination of a sacrificial feature to generate bubble flowforces. FIGS. 10A and 10B demonstrated the export of human hybridomacells from a sequestration pen 1002, having a connection region 1006 andan isolation region 1008. In this example, the sequestration pen did nothave a distinct or separate displacement force generation region.

The cells were cultured within sequestration pen 1002 of microfluidicdevice 1000 for three days (not shown). The export was performed byfocusing the laser (power was 90 milliwatt) with a 1.4 Ampere current onthe inner surface (thermal target) of the microfluidic circuit, whichwas a dielectrophoresis substrate and the cover comprising indium tinoxide (“ITO”) for 5-10 seconds. The specific location illuminated was adiscrete selected point 1020 of the inner surface at the base of thesequestration pen 1002, opposite the opening of the sequestration pen tothe microfluidic channel 1022. The thermal target was the substratewhich absorbed the optical illumination, converted the illumination tothermal energy, and thereby heated the surrounding fluidic medium. Inthis process, a portion of the substrate was destroyed, acting as asacrificial feature. The fluidic medium was heated sufficiently tonucleate a stream of gaseous bubbles. FIG. 10A showed a time pointduring the period of illumination as a bubble 1024 was formed at thebottom of the sequestration pen 1002 by focusing light on the substrate.The cells 1004 have been moved from the isolation region 1008 into theconnection region 1006 the sequestration pen. FIG. 10B showed thesequestration pen 1002 at a later time point, where a stream of bubbles1024 has grown in volume and the majority of cells 1004 have beenexported from the pen 1002 into the microfluidic channel 1022. Theexported cells 1004 were transferred to a wellplate, and singly seededfor further culturing. FIG. 10C shows one well of the wellplate after 3days, showing that the singly seeded cell was viable and had expanded.

Optical system for Examples 3 and 4. The optical train was modified toincorporate a 785 nm laser, Chroma ZT745spxrxt-UF1 dichroic filter(Chroma, Below Falls, VT), a ZET785nf (Chroma) emission filter, and a 4×Nikon objective.

Protocol for Examples 3 and 4. A 785 nm laser was focused on the innersurface (dielectrophoresis substrate) of a sequestration pen and coverabove the pen to produce 90 milliwatts (mW) of power for approximatelyone second, heating the fluidic medium and generating one or morebubbles. Cells were dislodged from respective isolation regions bybubble contact force and/or shear flow generated by the bubbles. Afterdislodgement, OET force was used to deliver and position individualdislodged cells into adjacent pens. The newly repositioned cells wereobserved after 24 hours in culture to determine effect on cell viabilityand proliferation.

Example 3

Optically driven displacement of OKT3 cells and viability. FIG. 11A-11Cdepict experimental results from before and after optically drivendisplacement and repositioning of OKT3 cells. FIG. 11A shows a series ofsequestration pens of microfluidic device 1100 prior to the export ofthe cells 1104 from sequestration pen 1102. Optical illumination wasdirected towards sacrificial feature 1120, a surface of thesequestration pen 1102, which acts as a thermal target. Resultingly,cells 1104 were dislodged. Single cells were positioned in adjacentsequestration pens. FIG. 11B showed the individual dislodged andrepositioned single cells 1104 b, 1104 c, 1104 d, and 1104 e in newlyoccupied pens after displacement and repositioning with OET. Originallyoccupied sequestration pen 1102 had a reduced number of cells 1104 a,which may have been dislodged but remained within sequestration pen1103. FIG. 11C showed the same sequestration pens 20 hours later. Asshown in FIG. 12C, cells 1104 b, 1104 c, 1104 d, and 1104 e continued todivide and proliferate after dislodging, export, and repositioning.Additionally, the remaining cells in originally occupied sequestrationpen 1102 also proliferated. These results indicated that the process ofoptical illumination, heating, dislodging and repositioning had nodetectable effect on cell viability in this experiment. As shown in FIG.12C the number of cells 1104 c having increased from two to four; thenumber of cells 1104 d increased from one to four, and cells 1104 eincreased from one to two.

Example 4 Optically Driven Displacement and Resultant Viability ofJIMT-1 Cells

Medium: Serum free medium (ThermoFisher Scientific, Cat. No. 12045-096),with a conditioning culture medium additive, B-27® Supplement (2% v/v).

FIGS. 12A-C depict experimental results from before and after opticallydriven displacement and repositioning of JIMT-1 cells (commerciallyavailable from AddexBio Cat. #C0006055), an adherent human breastcarcinoma cell line. FIG. 12A showed a series of sequestration pens ofmicrofluidic device 1200 prior to the displacement of cells 1204 fromsequestration pen 1202. Optical illumination was directed towardssacrificial feature 1220 (thermal target), a surface of thesequestration pen 1202, and cells 1204 were dislodged. FIG. 14B showedan individual cell 1204 a, dislodged and newly repositioned in an emptysequestration pen adjacent to originally occupied sequestration pen1202. FIG. 14C shows a timepoint 20 h after displacement andrepositioning showing that cells 1204 a were viable and have doubledfrom one cell to two, within the 20 h culture period.

Example 5

Induction of a cyclic (Marangoni effect) flow. FIGS. 13A to 13C depictexperimental results demonstrating a cyclic Marangoni-effect flow usinga cyclic culturing pen geometry. FIG. 13A shows a number ofmicro-objects 1305 (6 micron diameter polystyrene beads) at a first timepoint during which a laser was directed at thermal target 1320 to createa bubble 1330 which causes a Marangoni effect flow within the cyclicculturing pen 1302 and adjacent channel 1322 of microfluidic device1300. FIG. 13B shows the same cyclic culturing pen 1302 withmicro-objects 1305 at a second time point (after a bubble 1330 a brokeaway from the site of nucleation) during which the micro-objects 1305were cycled around counterclockwise (white arrow indicates flowdirection) through the culturing pen 1302 by the Marangoni-effect flow.FIG. 13C shows a third timepoint, during which laser illumination wasstill present. The micro-objects 1305 have pushed past the thermaltarget 1320, out into the channel 1322, and cycled back in to the secondside of the cyclic culturing pen 1302. In the experiment illustrated inFIGS. 13A to 13C, the bubble was nucleated by focusing a 785 laser ongold thermal targets deposited on the inner surface of the cover of themicrofluidic device. Specifically, the laser was used to generate a spotof light 40 microns in diameter and had 90 mW corresponding to: 1.4kW/cm{circumflex over ( )}2.

Example 6

Optically driven displacement of OKT3 cells, shorter period ofillumination. OKT3 murine hybridoma cells were cultured in fluidicmedium within sequestration pens of microfluidic device 1400 as shown.FIG. 14A shows a colony of cells 1440 maintained within the centralsequestration pen 1402. The group of cells are highlighted within thewhite oval, and no laser illumination has yet been introduced. FIG. 14Bshows the same group of cells 1440 (within the white oval) in thecentral sequestration pen 1402 of the figure, while the laser wasdirected at a discrete selected point 1420 (thermal target which is asacrificial feature) of the surface of the sequestration pen 1302 wheresome of the cells were contained, for a duration of time in a range ofabout 50 millisec to about 1000 millisec, using 1.4 amps. FIG. 14Cshowed that the resultant cavitation and collapse of a bubble nucleatedfrom the heat introduced by the laser illumination dislodged anddisplaced a group of cells 1404 b completely out of the sequestrationpen 1402, and into the microfluidic channel 1422. The remainder of thecells 1404 a, were somewhat displaced but were not been exported fromthe sequestration pen 1402.

Example 7

Optically driven displacement of OKT3 cells, longer period ofillumination. OKT3 murine hybridoma cells 1504 were maintained within afluidic medium within sequestration pens in microfluidic device 1500 andare shown, prior to any laser illumination in FIG. 15A, where the whiteoval points out the colony of cells to be dislodged. Laser illuminationis shown in FIG. 15B. The laser power was 1.4 amps, and the duration ofthe laser pulse was about 2000 millisec. The white oval surrounds thecells 1504 to be dislodged, and the discrete region of illumination 1520is at the bottom of the sequestration pen 1502, and particularly wasdirected at the microfluidic circuit material forming the sequestrationpen wall. The area targeted acted as a sacrificial feature (e.g.,thermal target). FIG. 15C illustrates the timepoint near the end of the2000 millisec laser illumination where the cells 1504 (within the whiteoval) were dislodged and were displaced towards the proximal opening ofthe sequestration pen to the flow region (e.g., microfluidic channel),pushed by a bubble under the group of cells 1504 (not visible).Blackening seen at the base of the sequestration pen shows destructionof the substrate material and some of the sequestration pen wallmaterial within the sequestration pen 1502 (See also post-illuminationsacrificial feature 1524, in FIG. 15D). FIG. 15D shows a time pointafter conclusion of optical illumination, where optically actuateddielectrophoretic forces were applied to continue moving the nowdislodged cells 1504 further out of the sequestration pen 1504. In FIG.15D, white bars 1530 1532, 1534, 1536 were the light (OET) patternsdisplayed upon the substrate surface, where the substrate included adielectrophoretic configuration. As the light pattern bars 1530 1532,1534, 1536 moved in the direction towards the opening of thesequestration pen to the microfluidic channel 1522, cells captured andrepelled by the dielectrophoretic force generated by each light barpattern, were moved toward the opening of the sequestration pen (see thecells 1504 b and 1504 c within the white ovals). Cells 1504 c, beingrepelled by light pattern bar 1530, were fully exported from thesequestration pen, and were re-located into the flow region (e.g., amicrofluidic channel 1522). FIG. 15E shows a later timepoint, when theoptically actuated dielectrophoretic light patterns 1530 1532, 1534,1536 completed a sequence of capturing, repelling and moving the cellsout of the sequestration pen, and fluidic flow was restored to the flowregion. Cells that had been dislodged by the laser pulse method andfurther moved out of the sequestration pen into the flow region wereexported out of the microfluidic device. Cells 1504 d (within white ovalfor emphasis) still remained in the sequestration pen but were clearlydislodged from their original position within the sequestration pen1502, prior to laser illumination (compare with FIG. 15A). Additionalsequences of optically actuated dielectrophoretic selecting and movingmay permit the remaining cells to be exported out of the sequestrationpen.

Recitation of Embodiments

1. A microfluidic device including an enclosure further including a flowregion and a sequestration pen, wherein the sequestration pen includes:a connection region, an isolation region and a displacement forcegeneration region, wherein: the connection region includes a proximalopening to the flow region and a distal opening to the isolation region,and the isolation region includes at least one fluidic connection to thedisplacement force generation region; and the displacement forcegeneration region further includes a thermal target.

2. The microfluidic device of claim 1, wherein the at least one fluidicconnection between the isolation region and the displacement forcegeneration region includes a cross sectional dimension configured toprevent passage of a micro-object from the isolation region to thedisplacement force generation region.

3. The microfluidic device of embodiment 1 or 2, wherein the at leastone fluidic connection between the isolation region and the displacementforce generation region includes a cross sectional dimension configuredto prevent fluidic flow from the displacement force generation region inthe absence of a force generated therein, except by diffusion.

4. The microfluidic device of any one of embodiments 1 to 3, wherein theat least one fluidic connection between the isolation region and thedisplacement force generation region includes one or more barriermodules, wherein the one or more barrier modules are configured toprevent passage of a micro-object from the isolation region to thedisplacement force generation region.

5. The microfluidic device of any one of embodiments 1 to 5, wherein thedisplacement force generation region further includes an opening to theflow region.

6. The microfluidic device of any one of embodiments 1-to 5, wherein thedisplacement force generation region has more than one fluidicconnection to the isolation region.

7. The microfluidic device of embodiment 6, wherein the sequestrationpen includes a cyclic flow path.

8. The microfluidic device of embodiment 7, wherein the cyclic flow pathincludes a constricted portion.

9. The microfluidic device of any one of embodiments 6 to 8, furtherincluding a second thermal target configured to produce a second cyclicflow of the fluidic medium upon optical illumination.

10. The microfluidic device of embodiment 9, wherein the first thermaltarget and the second thermal target are oriented to provide the firstcyclic flow and the second cyclic flow of the fluidic medium in oppositedirections.

11. The microfluidic device of any one of embodiments 1-5, wherein thedisplacement force generation region includes a single opening, whereinthe single opening is the fluidic connection to the isolation region.

12. The microfluidic device of any one of embodiments 1 to 11, whereinthe fluidic connection of the displacement force generation regionincludes a fluidic connector including at least one curved portion.

13. The microfluidic device of embodiment 12, wherein the at least onecurved portion of the fluidic connector includes a turn of about 60degrees to about 180 degrees.

14. The microfluidic device of embodiment 12 or 13, wherein the fluidicconnector of the displacement force generation region includes at leasttwo curved portions.

15. The microfluidic device of embodiment 14, wherein each of the atleast two curved portions of the fluidic connector includes a turn ofabout 60 degrees to about 180 degrees.

16. The microfluidic device of any one of embodiments 12 to 15, whereina width of the fluidic connector is the same as a width of the isolationregion and/or the displacement force generating region.

17. The microfluidic device of any one of embodiments 12- to 15, whereinthe fluidic connector includes a cross sectional dimension configured toprevent passage of a micro-object from the isolation region to thedisplacement force generation region.

18. The microfluidic device of any one of embodiments 1 to 17, whereinthe enclosure of the microfluidic device further includes a cover thatdefines, in part, the sequestration pen, wherein the thermal target isdisposed on the cover.

19. The microfluidic device of embodiment 18, wherein the thermal targetis disposed on an inner surface of the cover facing the enclosure.

20. The microfluidic device of any one of embodiments 1 to 17, whereinthe enclosure of the microfluidic device further includes a microfluidiccircuit structure that defines, in part, the sequestration pen, andwherein the thermal target is disposed on the microfluidic circuitstructure.

21. The microfluidic device of any one of embodiments 1 to 17, whereinthe enclosure of the microfluidic device further includes a base thatdefines, in part, the sequestration pen, and wherein the thermal targetis disposed on an inner surface of the base.

22. The microfluidic device of any one of embodiments 1 to 21, whereinthe thermal target includes a metal.

23. The microfluidic device of any one of embodiments 1 to 22, whereinthe thermal target has a contiguous shape.

24. The microfluidic device of any one of embodiments 1 to 22, whereinthe thermal target has a non-contiguous shape.

25. The microfluidic device of any one of embodiments 1 to 22 or 24,wherein the thermal target includes a plurality of microstructures.

26. The microfluidic device of any one of embodiments 1 to 21 or 23 to25, wherein the thermal target is a sacrificial feature.

27. The microfluidic device of any one of embodiments 1 to 26, whereinthe thermal target or the displacement force generation region isconfigured to constrain expansion of a gaseous bubble formed thereuponin one predominate direction.

28. The microfluidic device of any one of embodiments 1 to 27, whereinthe thermal target is positioned in a portion of the displacement forcegeneration region distal to the least one fluidic connection to theisolation region.

29. The microfluidic device of embodiment 28, wherein the displacementforce generation region has a width of approximately 20-100 microns.

30. The microfluidic device of any one of embodiments 1 to 29, whereinthe enclosure further includes a dielectrophoresis configuration.

31. The microfluidic device of embodiment 30, wherein thedielectrophoresis configuration is optically actuated.

32. The microfluidic device of any one of embodiments 1 to 31, whereinthe sequestration pen includes at least one surface that is a coatedsurface.

33. The microfluidic device of embodiment 32, wherein the coated surfaceis a covalently linked surface.

34. A microfluidic device including an enclosure including amicrofluidic circuit configured to contain a fluidic medium, wherein themicrofluidic circuit is configured to accommodate at least one cyclicflow of the fluidic medium; and a first thermal target disposed on asurface of the enclosure within the microfluidic circuit, wherein thefirst thermal target is configured to produce a first cyclic flow of thefluidic medium upon optical illumination.

35. The microfluidic device of embodiment 34, wherein the thermal targethas a contiguous shape.

36. The microfluidic device of embodiment 34, wherein the thermal targethas a pattern of shapes.

37. The microfluidic device of any one of embodiments 34 to 36, whereinthe thermal target includes a non-uniform thickness on the surface ofthe enclosure, wherein the non-uniform thickness is configured toprovide differential heating of the fluidic medium by the thermal targetupon optical illumination.

38. The microfluidic device of embodiment 36 or 37, wherein the patternof shapes is configured to provide differential heating of the fluidicmedium by the thermal target upon optical illumination.

39. The microfluidic device of any one of embodiments 34 to 38, whereinthe thermal target includes a metal.

40. The microfluidic device of any one of embodiments 34 to 39, whereinthe thermal target includes a plurality of microstructures.

41. The microfluidic device of embodiment 40, wherein the plurality ofmicrostructures includes a pattern of increasing density ofmicrostructures configured to provide differential heating of thefluidic medium by the thermal target upon optical illumination.

42. The microfluidic device of any one of embodiments 34 to 41, whereinthe enclosure of the microfluidic device further includes a microfluidicchannel and a sequestration pen, and further wherein the sequestrationpen is adjacent to and opens off of the microfluidic channel.

43. The microfluidic device of embodiment 42, wherein the cyclic flowpath includes a portion of the channel and at least a portion of thesequestration pen.

44. The microfluidic device of embodiment 42, wherein the sequestrationpen includes the cyclic flow path.

45. The microfluidic device of any one of embodiments 34 to 44, whereinthe cyclic flow path includes a constricted portion.

46. The microfluidic device of any one of embodiments 34 to 45, furtherincluding a second thermal target configured to produce a second cyclicflow of the fluidic medium upon optical illumination.

47. The microfluidic device of embodiment 46, wherein the first thermaltarget and the second thermal target are oriented to provide the firstcyclic flow and the second cyclic flow of the fluidic medium in oppositedirections.

48. The microfluidic device of embodiment 42, wherein the thermal targetis disposed on a surface within the microfluidic channel.

49. The microfluidic device of any one of embodiments 34 to 41, whereinthe enclosure of the microfluidic device further includes more than onemicrofluidic channel, wherein a first microfluidic channel is configuredto open from a second microfluidic channel at a first location along thesecond microfluidic channel and is further configured to reconnect tothe second microfluidic channel at a second location thereby forming themicrofluidic circuit; and the thermal target is disposed on a surfacewithin the first microfluidic channel.

50. The microfluidic device of embodiment 49, wherein at least onesequestration pen opens off of the first microfluidic channel.

51. The microfluidic device of embodiment 49 or 50, wherein a fluidicresistance of the first channel is approximately 10 to 100 times higherthan a fluidic resistance of the second channel.

52. The microfluidic device of any one of embodiments 49 to 51, whereinthe second microfluidic channel includes a width that is approximately1.5 to 3 times larger than a width of the first microfluidic channel.

53. The microfluidic device of embodiment 52, wherein the width of thesecond microfluidic channel is about 100 to 1000 microns.

54. The microfluidic device of any one of embodiments 49 to 53, whereinthe width of the first microfluidic channel is about 20 to 300 microns.

55. A microfluidic device including an enclosure including: amicrofluidic channel and a sequestration pen, and further wherein thesequestration pen is adjacent to and opens off of the microfluidicchannel and a thermal target is disposed in the channel adjacent to anopening to a sequestration pen, and wherein the thermal target isfurther configured to direct a flow of the fluidic medium into thesequestration pen upon optical illumination.

56. The microfluidic device of embodiment 55, wherein the thermal targetis disposed on a surface within the microfluidic channel.

57. The microfluidic device of embodiment 55 or 56, wherein the thermaltarget has a contiguous shape.

58. The microfluidic device of embodiment 55 or 56, wherein the thermaltarget has a pattern of shapes.

59. The microfluidic device of any one of embodiments 55 to 58, whereinthe thermal target includes a non-uniform thickness on the surface ofthe enclosure, wherein the non-uniform thickness is configured toprovide differential heating of the fluidic medium by the thermal targetupon optical illumination.

60. The microfluidic device of embodiment 58 or 59, wherein the patternof shapes is configured to provide differential heating of the fluidicmedium by the thermal target upon optical illumination.

61. The microfluidic device of any one of embodiments 55 to 60, whereinthe thermal target includes a metal.

62. The microfluidic device of any one of embodiments 55 to 60, whereinthe thermal target includes a plurality of microstructures.

63. The microfluidic device of embodiment 62, wherein the plurality ofmicrostructures includes a pattern of increasing density ofmicrostructures configured to provide differential heating of thefluidic medium by the thermal target upon optical illumination.

64. A kit for culturing micro-objects including: a microfluidic deviceof any one of embodiments 1-63; and, one or more reagents configured toprovide at least one coated surface within an enclosure of themicrofluidic device.

65. The kit of embodiment 64, further including at least one fluidicmedium.

66. A method of dislodging one or more micro-objects within amicrofluidic device, the method including the steps of: illuminating aselected discrete region containing or adjacent to one or moremicro-objects disposed within a fluidic medium in an enclosure of themicrofluidic device, wherein the enclosure includes a microfluidiccircuit including a flow region and a substrate; maintaining theillumination of the selected discrete region of a first period of timesufficient to generate a dislodging force, dislodging the one or moremicro-objects from the surface.

67. The method of embodiment 66, wherein the selected discrete regionhas an area of about 100 square microns.

68. The method of embodiment 66, wherein the selected discrete regionhas an area of about 25 square microns.

69. The method of embodiment 66, wherein the step of illuminatingincludes illuminating the selected discrete region with a laser.

70. The method of any one of embodiments 66 to 69, wherein the one ormore micro-objects are disposed upon a surface of the substrate.

71. The method of any one of embodiments 66 to 70, further including astep of maintaining the one or more micro-objects within the fluidicmedium in the enclosure for a second period of time before performingthe step of illuminating the selected discrete region.

72. The method of any one of embodiments 66 to 71, wherein the enclosureof the microfluidic device includes at least one sequestration pen.

73. The method of embodiment 72, wherein the one or more micro-objectsare disposed and/or maintained on a surface of the substrate within theat least one sequestration pen.

74. The method of any one of embodiments 66 to 73, wherein the step ofilluminating the selected discrete region includes illuminating withillumination having incident power in the range of about 1 mW to about1000 mW.

75. The method of embodiment 74, wherein the first period of time is inthe range from 10 microsec to 3000 millisec or 100 millisec to 3minutes.

76. The method of any one of embodiments 66 to 75, wherein the step ofilluminating includes illuminating at least one of the one or moremicro-objects.

77. The method of embodiment 76, wherein the first period of time is inthe range of 10 microsec to 200 millisec, thereby creating a cavitatingforce dislodging the one or more micro-objects.

78. The method of embodiment 76 or 77, wherein, when the one or moremicro-objects are disposed and/or maintained within the at least onesequestration pen, the selected discrete region is a location distal toa proximal opening of the sequestration pen to the flow region or at acentral location of the at least one sequestration pen.

79. The method of any one of embodiments 66 to 78, wherein the selecteddiscrete region is a selected point adjacent to the one or moremicro-objects.

80. The method of embodiment 79, wherein the step of illuminating theselected discrete region includes directing illumination towards thesubstrate; microfluidic circuit material of a wall; or a thermal target.

81. The method of embodiment 80, wherein the thermal target includes ametal deposit, a pattern of metal deposits, or microstructures patternedon a surface.

82. The method of embodiment 80 or 81, wherein the thermal targetincludes a sacrificial feature.

83. The method of any one of embodiments 79 to 82, wherein when the oneor more micro-objects are disposed within at least one sequestration penwithin the enclosure, the s selected discrete region is located near aproximal opening of the at least one sequestration pen to the flowregion.

84. The method of any one of embodiments 79 to 82, wherein when the oneor more micro-objects are disposed within at least one sequestration penwithin the enclosure, the selected discrete region includes a selectedpoint of microfluidic circuit material that helps to define the at leastone sequestration pen.

85. The method of any one of embodiments 79 to 82, wherein when the oneor more micro-objects are maintained within at least one sequestrationpen within the enclosure, the selected discrete region includes asacrificial feature disposed within the at least one sequestration pen.

86. The method of embodiment 85, wherein the sacrificial featureincludes microfluidic circuit material.

87. The method of any one of embodiments 79 to 86, wherein the firstperiod of time is in the range of about 10 microsec to 200 millisec.

88. The method of any one of embodiments 79 to 87, wherein the step ofilluminating the selected discrete region further includes heating afirst portion of the fluidic medium located within or adjacent to theselected discrete region, thereby creating a cavitating force.

89. The method of any one of embodiments 79 to 87, wherein the methodfurther includes: heating a first portion of the fluidic medium; andcreating a persistent gaseous bubble displacing a second portion of thefluidic medium surrounding the one or more micro-object.

90. The method of embodiment 89, wherein the step of displacing a secondportion of the fluidic medium further includes creating a cyclic fluidicflow of the fluidic medium during the first period of illumination.

91. The method of any one of embodiments 79 to 87, wherein the methodfurther includes: heating a first portion of the fluidic medium; andcreating one or more gaseous bubbles, thereby creating a shear flow ofthe fluidic medium towards the one or more micro-objects.

92. The method of any one of embodiments 79 to 87, wherein the methodfurther includes: heating a first portion of the fluidic medium;creating a plurality of gaseous bubbles configured to stream towards theone or more micro-objects; and contacting the one or more micro-objectswith a meniscus of at least one gaseous bubble of the plurality ofgaseous bubble.

93. The method of any one of embodiments 89 to 92, wherein the firstperiod of time is in the range of about 100 millisec to about 3000minutes.

94. The method of embodiment 91 or 92, wherein the first period is inthe range of about 1000 millisec to about 2000 millisec.

95. The method of any one of embodiments 79 to 94, wherein when the oneor more micro-objects are maintained within a sequestration pen withinthe enclosure, the selected discrete region includes at least a part ofa wall forming a distal end of the sequestration pen, wherein the wallis positioned opposite to a proximal opening to the flow region.

96. The method of any one of embodiments 79 to 94, wherein when the oneor more micro-objects are disposed within a sequestration pen within theenclosure, the selected discrete region is located in a displacementforce generation region of the sequestration pen.

97. The method of embodiment 96, wherein the one or more micro-objectsare disposed within an isolation region of the sequestration pen and thedisplacement force generation region is fluidically connected to theisolation region.

98. The method of any one of embodiments 66 to 93, further including astep of exporting the one or more micro-objects from at least onesequestration pen disposed within the enclosure.

99. The method of embodiment 98, wherein the step of exporting the oneor more micro-objects from the at least one sequestration pen includesmoving the one or more micro-objects with dielectrophoresis force.

100. The method of any one of embodiments 66 to 99, further including astep of exporting the one or more micro-objects from the flow region ofthe enclosure of the microfluidic device.

101. The method of embodiment 100, wherein the step of exporting the oneor more micro-objects from the flow region includes using fluidic flowor dielectrophoresis forces.

102. A method of mixing fluidic media, and/or micro-objects containedtherein, within an enclosure of a microfluidic device, the methodincluding: focusing a light source on a thermal target disposed on asurface of the enclosure within a microfluidic circuit including atleast one fluidic medium and/or micro-objects, thereby heating a firstportion of the at least one fluidic medium; and inducing a cyclic flowof the at least one fluidic medium within the microfluidic circuitthereby mixing the fluidic media and/or micro-objects disposed therein.

103. The method of embodiment 102, wherein the thermal target isdisposed within a first microfluidic channel which is configured tobranch off of a second fluidic channel at a first location and is alsoconfigured to rejoin the second fluidic channel at a second location,wherein the thermal target is disposed on the surface therebetween.

Although specific embodiments and applications of the disclosure havebeen described in this specification, these embodiments and applicationsare exemplary only, and many variations are possible.

The invention claimed is:
 1. A method of dislodging one or moremicro-objects within a microfluidic device, the method comprising thesteps of: illuminating a selected discrete region containing or adjacentto one or more micro-objects disposed within a fluidic medium in anenclosure of the microfluidic device, wherein the enclosure comprises amicrofluidic circuit comprising a flow region, a substrate, and at leastone sequestration pen comprising: a connection region; an isolationregion; and a displacement force generation region, wherein theconnection region comprises a proximal opening to the flow region and adistal opening to the isolation region and wherein a fluidic connectionbetween the isolation region and the displacement force generationregion is configured to prevent passage of a micro-object from theisolation region to the displacement force generation region; andmaintaining the illumination of the selected discrete region for a firstperiod of time sufficient to generate a dislodging force, dislodging theone or more micro-objects from a surface of the microfluidic device. 2.The method of claim 1, wherein the step of illuminating comprisesilluminating the selected discrete region with a laser.
 3. The method ofclaim 1, wherein the one or more micro-objects are disposed upon asurface of a substrate of the microfluidic device.
 4. The method ofclaim 3, wherein the one or more micro-objects are disposed and/ormaintained on a surface of the substrate within the at least onesequestration pen.
 5. The method of claim 1, wherein the first period oftime is in the range of about 10 microseconds to minutes, therebycreating a cavitating force dislodging the one or more micro-objects. 6.The method of claim 5, wherein, when the one or more micro-objects isdisposed and/or maintained within the at least one sequestration pen,the selected discrete region is a location distal to a proximal openingof the at least one sequestration pen to the flow region or is a centrallocation of the at least one sequestration pen.
 7. The method of claim1, wherein the selected discrete region is a selected point adjacent tothe one or more micro-objects.
 8. The method of claim 7, wherein thestep of illuminating the selected discrete region comprises directingillumination towards the substrate, microfluidic circuit material of awall of the microfluidic device, or a thermal target.
 9. The method ofclaim 7, wherein when the one or more micro-objects are disposed withinat least one sequestration pen, the selected discrete region comprises aselected point of microfluidic circuit material that helps define the atleast one sequestration pen.
 10. The method of claim 7, wherein the stepof illuminating the selected discrete region further comprises heating afirst portion of the fluidic medium located within or adjacent to theselected discrete region, thereby creating a cavitating force.
 11. Themethod of claim 7, wherein the method further comprises: heating a firstportion of the fluidic medium; and, creating a persistent gaseous bubbledisplacing a second portion of the fluidic medium surrounding the one ormore micro-objects.
 12. The method of claim 7, wherein the methodfurther comprises: heating a first portion of the fluidic medium; andcreating one or more gaseous bubbles, thereby creating a shear flow ofthe fluidic medium towards the one or more micro-objects.
 13. The methodof claim 7, wherein the method further comprises: heating a firstportion of the fluidic medium; creating a plurality of gaseous bubblesconfigured to stream towards the one or more micro-objects; andcontacting the one or more micro-objects with a meniscus of at least onegaseous bubble of the plurality of gaseous bubble.
 14. The method ofclaim 7, wherein, when the one or more micro-objects are maintainedwithin at least one sequestration pen, the selected discrete regioncomprises at least a part of a wall forming a distal end of the at leastone sequestration pen, wherein the wall is positioned opposite to aproximal opening to the flow region.
 15. The method of claim 7, whereinwhen the one or more micro-objects are disposed within at least onesequestration pen, the selected discrete region is located in adisplacement force generation region of the at least one sequestrationpen.
 16. The method of claim 15, wherein the one or more micro-objectsare disposed within an isolation region of the at least onesequestration pen and the displacement force generation region isfluidically connected to the isolation region.
 17. The method of claim1, further comprising a step of exporting the one or more micro-objectsfrom at least one sequestration pen disposed within the enclosure. 18.The method of claim 17, wherein the step of exporting the one or moremicro-objects from the at least one sequestration pen comprises movingthe one or more micro-objects with di electrophoresis force.
 19. Themethod of claim 1, further comprising a step of exporting the one ormore micro-objects from the flow region of the enclosure of themicrofluidic device.
 20. The method of claim 19, wherein the step ofexporting the one or more micro-objects from the flow region comprisesusing fluidic flow or dielectrophoresis forces.